Antibody fc and fab variants

ABSTRACT

The present invention relates to modified antibodies. In particular, the present invention relates to recombinant monoclonal antibodies or fragments, including chimeric, primatized or humanized antibodies or fragments, having reduced effector function and altered ability to mediate cell signaling activity by a target antigen. In addition, the present invention relates to nucleic acid molecules encoding such antibodies, and vectors and host cells comprising such nucleic acid molecules. The invention further relates to methods for producing the antibodies of the invention, and to methods of using these antibodies in treatment of disease.

FIELD OF THE INVENTION

The present invention relates to modified antibodies. In particular, thepresent invention relates to recombinant monoclonal antibodies orfragments, including chimeric, primatized or humanized antibodies orfragments, having reduced effector function and altered ability tomediate cell signaling activity by a target antigen. In addition, thepresent invention relates to nucleic acid molecules encoding suchantibodies, and vectors and host cells comprising such nucleic acidmolecules. The invention further relates to methods for producing theantibodies of the invention, and to methods of using these antibodies intreatment of disease.

BACKGROUND

Antibodies, also called immunoglobulins, have a basic structurecomprising four polypeptide chains: two identical heavy (H) chainspaired with two identical light (L) chains. Each heavy and light chaincomprises a variable region (VH and VL, respectively) and a constantregion (CH and CL, respectively). The CH region has 3 domains (CH1, CH2,and CH3), while the smaller CL region has only one domain (simplyreferred to as CL). Each VH and VL region comprises 3 complementaritydetermining regions (CDRs) flanked by 4 framework regions in thefollowing order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are the mostvariable part of the V region, and determine the antigen specificity ofthe antibody. Together, a paired VH and VL region form the antigenbinding site, and bivalent antibodies have two such antigen bindingsites.

The Fc region of an antibody, i.e., the terminal ends of the heavychains of antibody spanning domains CH2, CH3 and a portion of the hingeregion, is limited in variability and is involved in effecting thephysiological roles played by the antibody. The effector functionsattributable to the Fc region of an antibody vary with the class andsubclass of antibody and include binding of the antibody via the Fcregion to a specific Fc receptor (“FcR”) on a cell which triggersvarious biological responses.

These receptors typically have an extracellular domain that mediatesbinding to Fc, a membrane spanning region, and an intracellular domainthat may mediate some signaling event within the cell. The Fc receptorsare expressed in a variety of immune cells including monocytes,macrophages, neutrophils, dendritic cells, eosinophils, mast cells,platelets, B cells, large granular lymphocytes, Langerhans' cells,natural killer (NK) cells, and T cells. Formation of the Fc/FcγR complexrecruits these effector cells to sites of bound antigen, typicallyresulting in signaling events within the cells and important subsequentimmune responses such as release of inflammation mediators, B cellactivation, endocytosis, phagocytosis, and cytotoxic attack. The abilityto mediate cytotoxic and phagocytic effector functions is a potentialmechanism by which antibodies destroy targeted cells. The cell-mediatedreaction wherein nonspecific cytotoxic cells that express FcγRsrecognize bound antibody on a target cell and subsequently cause lysisof the target cell is referred to as antibody dependent cell-mediatedcytotoxicity (ADCC) (Ravetch, et al., Annu Rev Immunol 19 (2001)275-290). The cell-mediated reaction wherein nonspecific cytotoxic cellsthat express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell is referred to asantibody dependent cell-mediated phagocytosis (ADCP). In addition, anoverlapping site on the Fc region of the molecule also controls theactivation of a cell independent cytotoxic function mediated bycomplement, otherwise known as complement dependent cytotoxicity (CDC).

For the IgG class of antibodies, ADCC and ADCP are governed byengagement of the Fc region with a family of receptors referred to asFcγ receptors (FcγRs). In humans, this protein family comprises FcγRI(CD64); FcγRII (CD32), including isoforms FcγRIIA, FcγRIIB, and FcγRIIC;and FcγRIII (CD16), including isoforms FcγRIIIA and FcγRIIIB (Raghavan,and Bjorkman, Annu. Rev. Cell Dev. Biol. 12 (1996) 181-220; Abes, etal., Expert Reviews VOL 5(6), (2009) 735-747). FcγRs are expressed on avariety of immune cells, and formation of the Fc/FcγR complex recruitsthese cells to sites of bound antigen, typically resulting in signalingand subsequent immune responses such as release of inflammationmediators, B cell activation, endocytosis, phagocytosis, and cytotoxicattack. Furthermore, whereas FcγRI, FcγRIIA/c, and FcγRIIIA areactivating receptors characterized by an intracellular immunoreceptortyrosine-based activation motif (ITAM), FcγRIIB has an inhibition motif(ITIM) and is therefore inhibitory. Moreover, de Reys, et al., Blood,81, (1993) 1792-1800 concluded that platelet activation and aggregationinduced by monoclonal antibodies, like for example CD9, is initiated byantigen recognition followed by an Fc domain dependent step, whichinvolves the FcγRII-receptor (see also: Taylor, et al., Blood 96 (2000)4254-4260). While FcγRI binds monomelic IgG with high affinity, FcγRIIIand FcγRII are low-affinity receptors, interacting with complexed oraggregated IgG.

In many circumstances, the binding and stimulation of effector functionsmediated by the Fc region of immunoglobulins is highly beneficial,however, in certain instances effector function can lead to adverseeffects during antibody-mediated treatment of disease. This isparticularly true for those antibodies designed to deliver a drug (e.g.,toxins and isotopes) to the target cell where the Fc/FcγR mediatedeffector functions bring healthy immune cells into the proximity of thedeadly payload, resulting in depletion of normal lymphoid tissue alongwith the target cells. In other instances, for example, where blockingthe interaction of a widely expressed receptor with its cognate ligandis the objective, recruitment of immune effector cells results inunwanted toxicity. Also, in the instance where a therapeutic antibodyexhibited promiscuous binding across a number of human tissues inductionof effector function leads to adverse events and toxicity. Last but notleast, affinity to the FcγRII receptor can lead to platelet activationand aggregation via FcγRII receptor binding, which is a seriousside-effect of such antibodies.

In addition to mediating effector functions, monoclonal antibodies canmodulate cellular functions by inducing or inhibiting cell signalingpathways. For example, monoclonal antibodies have been shown to mediateantigen cross-linking, activate death receptors (e.g., by facilitatingoligomerization of receptors or mimicking ligand binding), and blockingof ligand-mediated cell signaling in cell growth differentiation, and/orproliferation pathways (see, e.g., Ludwig et al, Oncogene (2003) 22:9097-9106). Apoptosis, or programmed cell death, can be triggered byseveral different mechanisms. For example, the activation of signalingpathways through cell membrane-bound “death receptors”, e.g., members ofthe tumor necrosis factor receptor (TNFR) superfamily, can lead toinduction of direct cell death. Likewise, dimerization or cross-linkingof surface antigen, e.g., CD20, can also induce direct cell death (see,e.g., Ludwig et al, Oncogene (2003) 22: 9097-9106). The orientation ofthe variable domains of e.g. IgG type antibodies seem to play a crucialrole regarding antibody mediated induction of direct cell death.

The interface between the VH and CH1 domains comprises conserved aminoacids (see e.g., Lesk and Chothia, Nature (1988) 335(8):188-190). Thearea of contact can be described as a “molecular ball-and-socket joint”.This joint determines the “elbow motion” and also the so called “elbowangle” of the VH and VL regions with respect to the CH1 and CL regions,and prevents a rigid contact from forming between the V and C regions(Lesk and Chothia, Nature (1988) 335(8): 188-190)). The “socket” of thisball-and-socket joint is formed by amino acid residues in the VHframework region whereas the “ball” is formed by amino acid residues inthe CH1 domain. Differences in the amino acids at these positions candictate the elbow angle that is formed between the V and C regions, andtherefore the orientation of the VH-VL dimer (see Lesk and Chothia,Nature (1988) 335(8): 188-190). Conformational alterations of the elbowangle have been identified to be responsible for significant changes inbinding behavior of the antibody/antigen interaction without changingoverall affinity.

SUMMARY

Recognizing the tremendous therapeutic potential of modified antibodiesthat have reduced ability to induce effector functions and alteredability to induce direct cell death, the present inventors developedsuch antibodies, as well as a method for producing such antibodies.Inter alia, this method involves producing recombinant, chimericantibodies or chimeric fragments thereof.

There remains a need for monoclonal antibodies with improved therapeuticpotential for human therapy. Modulating induction of effector functionand enhancing signaling pathways with monoclonal antibodies is verychallenging, but modulating antigens associated with cell signaling,including, but not limited to, the induction of direct cell death whilecontrolling effector function, is much needed for improved cancertherapy. Unexpectedly, the present inventors found that the pointmutation Asn297Asp leads to reduced or abolished effector function butresidual ADCP function. The inventors further found that the mutationPro329Gly also leads to reduced or abolished effector function butresidual ADCP function and the Asn297Asp and Pro329Gly mutationsunexpectedly display a comparable and unique pattern of activation ofeffector functions. Surprisingly, this mutation can be combined withmutations in the elbow hinge part of the antibodies as disclosed herein,leading to altered induction of direct cell death. In combination, themodifications as disclosed herein allow modulation of induction ofdirect cell death and selective ablation of effector functions ascompared to non-modified parent antibodies.

Accordingly, one aspect of the invention is an antibody comprising avariant heavy chain region comprising at least one amino acidsubstitution relative to the parent non-substituted heavy chain region,leading to strongly reduced or abolished ADCC and CDC function, residualADCP function, reduced or abolished binding to Fc receptors, reduced orabolished binding to C1q and reduced or abolished toxicities. Anotheraspect of the invention is an antibody comprising a variant heavy chainregion comprising at least one amino acid substitution relative to theparent non-substituted heavy chain region, wherein the heavy chainregion of the parent non-substituted antibody comprises the amino acidresidue Asn297, and wherein said substitution is Asn297Asp. Anotheraspect of the invention is an antibody comprising a variant heavy chainregion comprising at least one amino acid substitution relative to theparent non-substituted heavy chain region, wherein the heavy chainregion of the parent non-substituted antibody comprises the amino acidresidue Pro329, and wherein said substitution is Pro329Gly. A furtheraspect of the invention is an antibody as described herein, whereininduction of effector function is reduced compared to effector functioninduced by an antibody comprising the parent non-substituted heavy chainregion.

In a specific aspect of the invention said parent non-substitutedantibody is an anti-CD20 antibody. In another specific aspect of theinvention the parent non-substituted antibody is a type I anti-CD20antibody. In yet another specific aspect of the invention the parentnon-substituted antibody is a type II anti-CD20 antibody. In a preferredaspect of the invention the parent non-substituted antibody isobinutuzumab. In another preferred aspect of the invention the parentnon-substituted antibody is rituximab.

Another aspect of the invention is an antibody as described herein,wherein FcγRIII binding by the antibody comprising the variant heavychain region is abolished compared to binding to FcγRIII by the parentnon-substituted antibody comprising asparagine at position 297. Yetanother aspect of the invention is an antibody as described herein,wherein ADCC function induced by the antibody comprising the variantheavy chain region is abolished or strongly reduced compared to ADCCfunction induced by the parent non-substituted antibody comprisingasparagine at position 297. Yet another aspect of the invention is anantibody as described herein, wherein FcγRI binding by the antibodycomprising the variant heavy chain region is reduced compared to bindingto FcγRI by the parent non-substituted antibody comprising asparagine atposition 297. Yet another aspect of the invention is an antibody asdescribed herein, wherein induction of ADCP function induced by theantibody comprising the variant heavy chain region is reduced comparedto ADCP function induced by the parent non-substituted antibodycomprising asparagine at position 297, wherein said antibody comprisingthe variant heavy chain retains residual ADCP function. Yet anotheraspect of the invention is an antibody as described herein, whereininduction of CDC function induced by the antibody comprising the variantheavy chain region is strongly reduced compared to CDC function inducedby the parent non-substituted antibody comprising asparagine at position297. Another aspect of the invention is an antibody as described hereinwherein the parent non-substituted antibody comprises the amino acidresidue Pro329, wherein FcγRIII binding by the antibody comprising thevariant heavy chain region is abolished compared to binding to FcγRIIIby the parent non-substituted antibody comprising proline at position329. Yet another aspect of the invention is an antibody as describedherein, wherein ADCC function induced by the antibody comprising thevariant heavy chain region is abolished or strongly reduced compared toADCC function induced by the parent non-substituted antibody comprisingproline at position 329. Yet another aspect of the invention is anantibody as described herein, wherein FcγRI binding by the antibodycomprising the variant heavy chain region is reduced compared to bindingto FcγRI by the parent non-substituted antibody comprising proline atposition 329. Yet another aspect of the invention is an antibody asdescribed herein, wherein induction of ADCP function induced by theantibody comprising the variant heavy chain region is reduced comparedto ADCP function induced by the parent non-substituted antibodycomprising proline at position 329, wherein said antibody comprising thevariant heavy chain retains residual ADCP function. Yet another aspectof the invention is an antibody as described herein, wherein inductionof CDC function induced by the antibody comprising the variant heavychain region is strongly reduced compared to CDC function induced by theparent non-substituted antibody comprising proline at position 329.

Yet another aspect of the invention is an antibody as described herein,wherein the variant heavy chain region comprises a further amino acidsubstitution relative to the parent non-substituted heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residue Pro151, and wherein said furthersubstitution is at said amino acid residue Pro151, wherein direct celldeath induced by the antibody comprising the variant heavy chain regionis altered compared to direct cell death induced by the antibodycomprising proline at position 151. Another aspect of the invention isthe antibody as described herein, wherein direct cell death induced bythe antibody comprising the variant heavy chain region is increasedcompared to direct cell death induced by the antibody comprising prolineat position 151. Another aspect of the invention is the antibody asdescribed herein, wherein direct cell death induced by the antibodycomprising the variant heavy chain region is decreased compared todirect cell death induced by the antibody comprising proline at position151.

Another aspect of the invention is the antibody as described herein,wherein Pro151 is substituted with an amino acid selected from the groupconsisting of alanine and phenylalanine. A specific aspect to theinvention is the antibody described herein, wherein Pro151 issubstituted with phenylalanine. Another specific aspect to the inventionis the antibody described herein, wherein Pro151 is substituted withalanine.

Yet another aspect of the invention is an antibody as described herein,wherein the variant heavy chain region comprises a further amino acidsubstitution relative to the parent non-substituted heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residue Val11, and wherein said furthersubstitution is at said amino acid residue Val11, wherein direct celldeath induced by the antibody comprising the variant heavy chain regionis altered compared to direct cell death induced by the antibodycomprising valine at position 11. Another aspect of the invention is theantibody as described herein, wherein direct cell death induced by theantibody comprising the variant heavy chain region is increased comparedto direct cell death induced by the antibody comprising valine atposition 11. Another aspect of the invention is the antibody asdescribed herein, wherein direct cell death induced by the antibodycomprising the variant heavy chain region is decreased compared todirect cell death induced by the antibody comprising valine at position11.

Another aspect of the invention is the antibody as described herein,wherein Val11 is substituted with an amino acid selected from the groupconsisting of alanine, glycine, phenylalanine, threonine and tryptophan.A specific aspect of the invention is the antibody as described herein,wherein Val11 is substituted with an amino acid selected from the groupconsisting of phenylalanine, threonine and tryptophan. Another specificaspect of the invention is the antibody as described herein, whereinVal11 is substituted with an amino acid selected from the groupconsisting of alanine and glycine.

Yet another aspect of the invention is an anti-CD20 antibody comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residues Asn297and Pro151, said variant heavy chain region comprising an amino acidsubstitution relative to the parent non-substituted heavy chain region,wherein said substitution is Asn297Asp, wherein induction of effectorfunction is reduced compared to effector function induced by an antibodycomprising the parent non-substituted heavy chain region, said variantheavy chain region comprising at least one further amino acidsubstitution at position Pro151 in the heavy chain, wherein direct celldeath induced by the antibody comprising the variant heavy chain regionis altered compared to direct cell death induced by the antibodycomprising proline at position 151. Yet another aspect of the inventionis an anti-CD20 antibody comprising a variant heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residues Pro329 and Pro151, said variant heavychain region comprising an amino acid substitution relative to theparent non-substituted heavy chain region, wherein said substitution isPro329Gly, wherein induction of effector function is reduced compared toeffector function induced by an antibody comprising the parentnon-substituted heavy chain region, said variant heavy chain regioncomprising at least one further amino acid substitution at positionPro151 in the heavy chain, wherein direct cell death induced by theantibody comprising the variant heavy chain region is altered comparedto direct cell death induced by the antibody comprising proline atposition 151.

Yet another aspect of the invention is an anti-CD20 antibody comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residues Asn297and Val11, said variant heavy chain region comprising an amino acidsubstitution relative to the parent non-substituted heavy chain region,wherein said substitution is Asn297Asp, wherein induction of effectorfunction is reduced compared to effector function induced by an antibodycomprising the parent non-substituted heavy chain region, said variantheavy chain region comprising at least one further amino acidsubstitution at position Val11 in the heavy chain, wherein direct celldeath induced by the antibody comprising the variant heavy chain regionis altered compared to direct cell death induced by the antibodycomprising valine at position 11. Yet another aspect of the invention isan anti-CD20 antibody comprising a variant heavy chain region, whereinthe heavy chain region of the parent non-substituted antibody comprisesthe amino acid residues Pro329 and Val11, said variant heavy chainregion comprising an amino acid substitution relative to the parentnon-substituted heavy chain region, wherein said substitution isPro329Gly, wherein induction of effector function is reduced compared toeffector function induced by an antibody comprising the parentnon-substituted heavy chain region, said variant heavy chain regioncomprising at least one further amino acid substitution at positionVal11 in the heavy chain, wherein direct cell death induced by theantibody comprising the variant heavy chain region is altered comparedto direct cell death induced by the antibody comprising valine atposition 11.

Another aspect of the invention is the antibody as described herein,wherein the antibody specifically binds to CD20. In a specific aspect ofthe invention, the antibody binds to CD20 with a dissociation constant(Kd) on cells of 10 nM or less as determined by scatchard analysis.

Another aspect of the invention is a polynucleotide encoding a variantheavy chain region of an antibody as described herein. Another aspect ofthe invention is a polynucleotide encoding a light chain region of anantibody as described herein. Yet another aspect of the invention is avector comprising at least one of the polynucleotides as describedherein. Another aspect of the invention is a polycistronic vectorcomprising the polynucleotides as described herein. Another aspect ofthe invention is a host cell comprising the vector or a polynucleotideas described herein.

Another aspect of the invention is a method for the production of anantibody as described herein comprising (i) culturing the host cell asdescribed herein under conditions permitting the expression of saidpolynucleotide; and (ii) recovering said antibody from the culturemedium.

Another aspect of the invention is a pharmaceutical compositioncomprising an antibody as described herein and a pharmaceuticallyacceptable carrier. Yet another aspect of the invention is an antibodyas described herein for use as a medicament. Yet another aspect of theinvention is an antibody as described herein for use in treating adisease selected from the group consisting of proliferative disorder andautoimmune disease.

Another aspect of the invention is an antibody as described herein foruse in treating a proliferative disorder as described herein,characterized in that said proliferative disorder is a CD20 expressingcancer. Yet another aspect of the invention is an antibody as describedherein for use in treating a CD20 expressing cancer, characterized inthat said cancer is selected from the group consisting of lymphoma andlymphocytic leukemia. Yet another aspect of the invention is an antibodyas described herein for use in treating an autoimmune disease asdescribed herein, characterized in that said autoimmune disease isselected from the group consisting of rheumatoid arthritis, lupus,multiple sclerosis, Sjögren's syndrome and transplant rejection.

Another aspect of the invention is a method for treating a diseaseselected from the group consisting of proliferative disorder andautoimmune disease comprising administering to an individual aneffective amount of the antibody as described herein. Yet another aspectof the invention is a method of treating an individual having cancercomprising administering to the individual an effective amount of theantibody as described herein, wherein said proliferative disorder is aCD20 expressing cancer. Yet another aspect of the invention is a methodof treating an individual having cancer comprising administering to theindividual an effective amount of the antibody as described herein,wherein said cancer is selected from the group consisting of lymphomaand lymphocytic leukemia. Yet another aspect of the invention is amethod of treating an individual having an autoimmune disease comprisingadministering to the individual an effective amount of the antibody asdescribed herein, wherein said autoimmune disease is selected from thegroup consisting of rheumatoid arthritis, lupus, multiple sclerosis,Sjögren's syndrome and transplant rejection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Induction of cell death by CD20 antibody (obinutuzumab, rituximab) Fcvariants as measured by Annexin V binding and PI staining was determinedusing CD20-expressing mantle cell lymphoma (Z-138). Fc variants(wildtype, glycoenineered (GE), P329G L234A L235A, N297D) were testedusing antibody concentrations from 16 ng/ml to 10 μg/ml for 21 hours.

FIGS. 2A-2D

Induction of ADCC by CD20 antibody (obinutuzumab, rituximab) Fc variantsas measured by tumor cell lysis after 4 hours of incubation with theantibody Fc variants (wildtype, glycoenineered (GE), P329G L234A L235Aand N297D),

FIG. 2A) Lysis of SU-DHL-4 cells as induced by peripheral bloodmononuclear cells (PBMCs) in the presence of antibody Fc variantsmeasured by FACS analysis

FIG. 2B) Lysis of Z-138 cells as induced by PBMCs in the presence ofantibody Fc variants measured by FACS analysis

FIG. 2C) Degranulation of natural killer cell (NK) after coculture withSU-DHL-4 cells in the presence of antibody Fc variants as assessed bybinding of anti-CD107a and flow cytometry

FIG. 2D) Degranulation of natural killer cells (NK) after coculture withZ-138 cells in the presence of antibody Fc variants as assessed bybinding of anti-CD107a and flow cytometry

FIGS. 3A-3D

CDC-dependent lysis of CD20 expressing tumor cells induced by CD20(obinutuzumab, rituximab) Fc variants was measured after incubation withthe antibody Fc variants (wildtype, glycoenineered (GE), P329G L234AL235A and N297D) in the presence of rabbit complement.

FIG. 3A) Tumor cell lysis after 2 hours of incubation of SU-DHL-4 cellswith rabbit complement in the presence of antibody Fc variants

FIG. 3B) Tumor cell lysis after 2 hours of incubation of Z-138 cellswith rabbit complement in the presence of antibody Fc variants

FIG. 3C) Tumor cell lysis after 22 hours of incubation of SU-DHL-4 cellswith rabbit complement in the presence of antibody Fc variants

FIG. 3D) Tumor cell lysis after 22 hours of incubation of Z-138 cellswith rabbit complement in the presence of antibody Fc variants

FIGS. 4A and 4B

B cell depletion in whole blood from healthy donors induced byobinutuzumab and rituximab Fc variants (wildtype, glycoenineered (GE),P329G L234A L235A and N297D) was measured by counting CD20/CD19 positivecells. Human whole blood was incubated with the antibody variants for 20hours.

FIG. 4A) B cell depletion as measured by FACS analysis in whole bloodfrom Donor 1

FIG. 4B) B cell depletion as measured by FACS analysis in whole bloodfrom Donor 2

FIGS. 5A-5D

ADCP induced by obinutuzumab and rituximab Fc variants (wildtype,glycoenineered (GE) and N297D) in the presence of human M1 or M2ceffector cells was measured by FACS analysis after co-incubation withtarget cells.

FIG. 5A) ADCP after 4 hours of incubation of SU-DHL-4 cells with humanM1 effector cells in the presence of antibody Fc variants

FIG. 5B) ADCP after 4 hours of incubation of Z-138 cells with human M1effector cells in the presence of antibody Fc variants

FIG. 5C) ADCP after 4 hours of incubation of SU-DHL-4 cells with humanM2c effector cells in the presence of antibody Fc variants

FIG. 5D) ADCP after 4 hours of incubation of Z-138 cells with human M2ceffector cells in the presence of antibody Fc variants

FIG. 6

Tumor volume development and tumor growth inhibition (TGI) values afterinduction of SC SU-DHL-4 tumors in SCID beige mice until day 49 weremeasured. Monotherapy treatment using obinutuzumab and rituximab Fcvariants (wildtype, glycoenineered (GE), P329G L234A L235A, N297D(aglyco)) started at day 21 after tumor cell inoculation and continuedadministered as once weakly dose for 4 weeks.

FIGS. 7A-7D

FcγRI binding as measured on a Biacore T100 system (GE Healthcare) withimmobilized anti-His capturing antibody.

FIG. 7A) FcγRI binding as compared between Herceptin, P-Selectin andfive different concentrations of the obinutuzumab variant N297D (aglyco)

FIG. 7B) FcγRI binding as compared between Herceptin, P-Selectin andobinutuzumab N297D

FIG. 7C) FcγRI binding and steady state affinity of the obinutuzumabvariant N297D (aglyco)

FIG. 7D) FcγRI binding as compared between different human IgG variants

FIGS. 8A-8C

FcγRIII binding as measured on a Biacore T100 system (GE Healthcare)with immobilized anti-His capturing antibody.

FIG. 8A) FcγRIII binding as compared between Herceptin, P-Selectin andfive different concentrations of the obinutuzumab (GA101) variant N297D(aglyco)

FIG. 8B) FcγRIII binding as compared for five different concentrationsof the obinutuzumab (GA101) variant N297D (aglyco)

FIG. 8C) FcγRIII binding as compared between different human IgGvariants

FIGS. 9A and 9B

FcγRII binding as measured on a Biacore T100 system (GE Healthcare) withimmobilized anti-His capturing antibody.

FIG. 9A) FcγRII binding as compared between different human IgG variants

FIG. 9B) FcγRII binding as compared between different human IgG variants

FIGS. 10A-10D

Induction of direct cell death by CD20 antibody (obinutuzumab, GA101)variants as measured by Annexin V binding and PI staining was determinedusing CD20-expressing mantle cell lymphoma (Z-138).

FIG. 10A) GA101 variants (GA101-wildtype, GA101-V11A, GA101-V11G,GA101-V11T, GA101-V11F, GA101-V11W, GA101-Ser114del, GA101-Ser114ins,GA101-P151A, GA101-LC 108, GA101-P151F, GA101-hinge ins, GA101-hingedel, GA101-P329F) were tested at an antibody concentration of 10 μg/ml;

FIG. 10B) GA101 variants (GA101-wildtype, GA101-V11A, GA101-V11G,GA101-V11T, GA101-V11F, GA101-V11W, GA101-Ser114del, GA101-Ser114ins,GA101-P151A, GA101-LC 108, GA101-P151F, GA101-hinge ins, GA101-hingedel, GA101-P329F) were tested at an antibody concentration of 0.1 μg/ml;

FIG. 10C) GA101 Fab variants (GA101-wildtype, GA101-V11F andGA101-P151F) and combined Fab/Fc variants (GA101-P329G L234A L235A,GA101-V11F P329G L234A L235A, GA101-P151F P329G L234A L235A andGA101-hinge del P329G L234A L235A) were compared for induction of directcell death;

FIG. 10D) Fab variants (V11F, P151F and hinge del) and combined Fabvariants (V11F P151F, P151F hinge del and V11F P151F hinge del) both ona GA101 P329G L234A L235A backbone were compared for induction of directcell death.

FIG. 11

B cell depletion induced by obinutuzumab variants (GA101-wildtype,GA101-P329G L234A L235A, GA101-V11F P329G L234A L235A and GA101-P151FP329G L234A L235A) was measured by counting CD20/CD19 positive cells.Human whole blood was incubated with antibody variants at differentconcentrations and for one or two days, respectively.

FIG. 12

Concentration-time profile for GA101-V11F P329G L234A L235A, GA101-P151FP329G L234A L235A and GA101-P329G L234A L235A in SCID beige mice.

FIGS. 13A and 13B

Melting temperature profile for GA101-N297D (aglyco), GA101-P329G,GA201-N297D (aglyco) and GA201-P329G as measured by Tryptophanfluorescence.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

Terms are used herein as generally used in the art, unless otherwisedefined as follows and herein.

The term “heavy chain”, “heavy chain domain” and “heavy chain region”are used interchangeably herein and refer to a polypeptide chain whichessentially consists of the heavy chain of an immunoglobulin heavy chainor fragments thereof retaining the same functionality compared to theimmunoglobulin heavy chain. In the present specification and claims, thenumbering of the residues in an immunoglobulin heavy chain is that ofKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991), expressly incorporated herein by reference in its entirety.Kabat et al. defined a numbering system for variable domain sequencesthat is applicable to any antibody. One of ordinary skill in the art canunambiguously assign this system of amino acid residue numbering to anyheavy chain region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) for variable domainsequences. Unless stated otherwise herein, references to residue number11 of a heavy chain region means residue numbering by the Kabatnumbering system (Kabat numbering). The “EU numbering” system or “EUindex as in Kabat” can be used when referring to a residue in animmunoglobulin heavy chain constant region. The EU index as in Kabatrefers to the residue numbering of the human IgG1 EU antibody. Thenumbering of residues can be determined for a given antibody byalignment at regions of homology of the sequence of the antibody with a“standard” Kabat numbered sequence. Unless stated otherwise herein,references to residue numbers 151 and 297 means residue numbering by theEU numbering system (EU numbering) set forth by Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Examples of twoanti-CD20 antibodies numbered according to Kabat (Kabat numbering or EUnumbering), in particular positions 11 (Kabat numbering), 151 (EUnumbering) and 297 (EU numbering) are included herein (SEQ ID NO: 01,SEQ ID NO: 02). Unless stated otherwise herein, references to residuenumber 329 means residue numbering by the EU numbering system.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those disclosed herein. Specific illustrative embodimentsfor measuring binding affinity are disclosed herein.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor its antigen.

As used herein, the term “agonist activity” is intended to refer toactivity of an agent (e.g., an antigen binding molecule) when itinteracts with (for example, binds to) a molecule associated with a cellsurface and initiates or induces a reaction.

As used herein, the term “altered cell signaling activity” is intendedto refer to an increase or decrease in the ability of an antibody toinduce or inhibit cell signaling activity of a target antigen.

As used herein, the term “altered cross-linking of one or more targetantigens” is intended to refer to an increase or decrease in the abilityof an antibody to bring into closer proximity to each other, and/or intocloser proximity with other membrane-associated molecules, and/or into amore favorable conformation for interaction target antigens that arecapable of forming complexes (e.g., through cross-linking of proteins,or oligomerization of membrane-associated receptors) to initiate cellsignaling activity.

As used herein, the term “altered induction of direct cell death” isintended to refer to an increase or decrease in the ability of anantibody to induce direct cell death.

As used herein, the term “altered induction of effector function” or“altered effector function” is intended to refer to an increase ordecrease in the ability of an antibody to induce effector function.

As used herein, “amino acid substitution” is intended to refer toreplacing one or more amino acids in a reference sequence (e.g., aparent molecule, such as an antibody). In one embodiment, amino acidsubstitution can be achieved by, for example, a point mutation in thesequence of a nucleic acid encoding a polypeptide as compared to aparent non-substituted sequence. In another embodiment, substitution ofan amino acid residue can be achieved by replacing the entire frameworkregion of the parent polypeptide with, for example, an Fc regionsequence that comprises the desired amino acid at the position to besubstituted in reference to the parent.

As used herein, the term “antagonist activity” is intended to refer toactivity of an agent (e.g., an antigen binding molecule) when itinteracts with (for example, binds to) a molecule on a cell and preventsinitiation or induction of a reaction or discontinues an ongoingreaction.

As used herein, the term “antibody” is intended to include wholeantibody molecules, including monoclonal, polyclonal and multispecific(e.g., bispecific) antibodies, as well as antibody fragments retainingbinding specificity, and fusion proteins that include a regionequivalent to the heavy chain region of an immunoglobulin and thatretain binding specificity. Also encompassed are “antibody fragments”that retain binding specificity including, but not limited to, VHfragments, VL fragments, Fab fragments, F(ab′)2 fragments, scFvfragments, Fv fragments, minibodies, diabodies, triabodies, andtetrabodies (see, e.g., Hudson and Souriau, Nature Med. 9: 129-134(2003), hereby incorporated by reference in its entirety). Alsoencompassed are humanized, primatized and chimeric antibodies. As usedherein, “whole antibody” refers to an immunoglobulin molecule comprisingtwo “heavy chains” and two “light chains”, each of which comprises avariable and constant region. As used herein, the term “modifiedantibody” is intended to refer to an antibody comprising at least oneamino acid residue substitution in the heavy chain variable regionand/or CH1 region and/or at least one amino acid residue substitution inthe light chain variable region and/or CL region and/or at least oneamino acid substitution in the Fc region.

An antibody “which binds” an antigen of interest, e.g. atumor-associated polypeptide antigen target, is one that binds theantigen with sufficient affinity such that the antibody is useful as atherapeutic agent in targeting a cell or tissue expressing the antigen,and does not significantly cross-react with other proteins. In suchembodiments, the extent of binding of the antibody to a “non-target”protein will be less than about 10% of the binding of the antibody toits particular target protein as determined by fluorescence activatedcell sorting (FACS) analysis or radioimmunoprecipitation (RIA). Withregard to the binding of an antibody to a target molecule, the term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetmeans binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule, which generally is a molecule of similar structure that doesnot have binding activity. For example, specific binding can bedetermined by competition with a control molecule that is similar to thetarget, for example, an excess of non-labeled target. In this case,specific binding is indicated if the binding of the labeled target to aprobe is competitively inhibited by excess unlabeled target. The term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetas used herein can be exhibited, for example, by a molecule having a Kdfor the target of about 10⁻⁴ M or less, alternatively about 10⁻⁵M orless, alternatively about 10⁻⁶ M or less, alternatively about 10⁻⁷ M orless, alternatively about 10⁻⁸ M or less, alternatively about 10⁻⁹ M orless, alternatively about 10⁻¹⁰ M or less, alternatively about 10⁻¹¹ Mor less, alternatively about 10⁻¹² M or less, or less. In oneembodiment, the term “specific binding” refers to binding where amolecule binds to a particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

The terms “anti-CD20 antibody” and “an antibody that specifically bindsto CD20” refer to an antibody that is capable of binding CD20 withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting CD20. In one embodiment, theextent of binding of an anti-CD20 antibody to an unrelated, non-CD20protein is less than about 10% of the binding of the antibody to CD20 asmeasured by a radioimmunoassay (RIA). In certain embodiments, anantibody that binds to CD20 has a dissociation constant (Kd) of ≤1 μM,≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M orless, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M) asmeasured by scatchard analysis. In certain embodiments, an anti-CD20antibody binds to an epitope of CD20 that is conserved among CD20 fromdifferent species. Depending on binding properties and biologicalactivities of anti-CD20 antibodies to the CD20 antigen, two types ofanti-CD20 antibodies (type I and type II anti-CD20 antibodies) can bedistinguished according to Cragg, M. S., et al., Blood 103 (2004)2738-2743; and Cragg, M. S., et al., Blood 101 (2003) 1045-1052, seeTable 1.

TABLE 1 Properties of Type I and Type II anti-CD20 Antibodies Type Ianti-CD20 antibodies Type II anti-CD20 antibodies Type I CD20 epitopeType II CD20 epitope Localize CD20 to lipid rafts Do not localize CD20to lipid rafts Increased CDC (if IgG1 isotype) Decreased CDC (if IgG1isotype) ADCC activity (if IgG1 isotype) ADCC activity (if IgG1 isotype)Full binding capacity Reduced binding capacity Homotypic aggregationStronger homotypic aggregation

One essential property of type I and type II anti-CD20 antibodies istheir mode of binding. Thus type I and type II anti-CD20 antibody can beclassified by the ratio of the binding capacities to CD20 on Raji cells(ATCC-No. CCL-86) of said anti-CD20 antibody compared to rituximab.

An antibody which “induces direct cell death” or “induces apoptosis” or“induces apoptosis-like direct cell death” is one which inducesprogrammed cell death as determined by binding of annexin V, cellshrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/orformation of membrane vesicles (called apoptotic bodies). The cell isusually one which expresses a CD20 polypeptide. Preferably the cell is atumor cell. More preferably the cell is a hematopoietic cell, such as amalignant B cell or a B cell involved in autoimmunity. Various methodsare available for evaluating the cellular events associated with directcell death. For example, phosphatidyl serine (PS) translocation can bemeasured by annexin binding.

An antibody which “induces cell death” is one which causes a viable cellto become nonviable. The cell is one which expresses a CD20 polypeptideand is of a cell type which specifically expresses or overexpresses aCD20 polypeptide. The cells may be cancerous or normal cells of theparticular cell type. The cell may be a normal B cell involved inautoimmunity. The cell may be a cancer cell. Preferably the cell is amalignant B cell. Cell death in vitro may be determined in the absenceof complement and immune effector cells to distinguish cell deathinduced by effector function. Thus, the assay for cell death may beperformed using heat inactivated serum (i.e., in the absence ofcomplement) and in the absence of immune effector cells. To determinewhether the antibody is able to induce cell death, loss of membraneintegrity as evaluated by uptake of propidium iodide (PI), trypan blue(see Moore et al. Cytotechnology 17:1-11 (1995)) or 7-AAD can beassessed relative to untreated cells.

By “antibody having altered antibody-dependent cell-mediatedcytotoxicity” (“ADCC”) is meant an antibody, as that term is definedherein, having altered ADCC as determined by any suitable method knownto those of ordinary skill in the art. One accepted in vitro ADCC assayis as follows:

1) the assay uses target cells that are known to express the targetantigen recognized by the antigen-binding region of the antibody;2) the assay uses human peripheral blood mononuclear cells (PBMCs),isolated from blood of a randomly chosen healthy donor, as effectorcells;3) the assay is carried out according to the following protocol:i) the PBMCs are isolated using standard density centrifugationprocedures and are suspended at 5×10⁶ cells/ml in RPMI cell culturemedium;ii) the target cells are grown by standard tissue culture methods,harvested from the exponential growth phase with a viability higher than90%, washed in RPMI cell culture medium, labeled with 100 micro-Curiesof ⁵¹Cr, washed twice with cell culture medium, and resuspended in cellculture medium at a density of 10⁵ cells/ml;iii) 100 microliters of the final target cell suspension above aretransferred to each well of a 96-well microtiter plate;iv) the antibody is serially-diluted from 4000 ng/ml to 0.04 ng/ml incell culture medium and 50 microliters of the resulting antibodysolutions are added to the target cells in the 96-well microtiter plate,testing in triplicate various antibody concentrations covering the wholeconcentration range above;v) for the maximum release (MR) controls, 3 additional wells in theplate containing the labeled target cells, receive 50 microliters of a2% (V/V) aqueous solution of non-ionic detergent (Nonidet, Sigma, St.Louis), instead of the antibody solution (point iv above);vi) for the spontaneous release (SR) controls, 3 additional wells in theplate containing the labeled target cells, receive 50 microliters ofRPMI cell culture medium instead of the antibody solution (point ivabove);vii) the 96-well microtiter plate is then centrifuged at 50×g for 1minute and incubated for 1 hour at 4° C.;viii) 50 microliters of the PBMC suspension (point i above) are added toeach well to yield an effector:target cell ratio of 25:1 and the platesare placed in an incubator under 5% CO₂ atmosphere at 37° C. for 4hours;ix) the cell-free supernatant from each well is harvested and theexperimentally released radioactivity (ER) is quantified using a gammacounter;x) the percentage of specific lysis is calculated for each antibodyconcentration according to the formula (ER-MR)/(MR-SR)×100, where ER isthe average radioactivity quantified (see point ix above) for thatantibody concentration, MR is the average radioactivity quantified (seepoint ix above) for the MR controls (see point v above), and SR is theaverage radioactivity quantified (see point ix above) for the SRcontrols (see point vi above);4) “increased ADCC” is defined as either an increase in the maximumpercentage of specific lysis observed within the antibody concentrationrange tested herein, and/or a reduction in the concentration of antibodyrequired to achieve one half of the maximum percentage of specific lysisobserved within the antibody concentration range tested herein. Theincrease in ADCC is relative to the ADCC, measured with the above assay,mediated by the same antibody, produced by the same type of host cells,using the same standard production, purification, formulation andstorage methods, which are known to those skilled in the art, but thathas not been modified by amino acid substitution as disclosed herein orglycoengineering. Thus, “decreased ADCC” is defined as either a decreasein the maximum percentage of specific lysis observed within the antibodyconcentration range tested herein, and/or an increase in theconcentration of antibody required to achieve one half of the maximumpercentage of specific lysis observed within the antibody concentrationrange tested herein. The decrease in ADCC is relative to the ADCC,measured with the above assay, mediated by the same antibody, producedby the same type of host cells, using the same standard production,purification, formulation and storage methods, which are known to thoseskilled in the art, but that has not been modified by amino acidsubstitution as disclosed herein or glycoengineering.

As used herein, the term “apoptosis” is intended to refer to programmedcell death, which is characterized by certain cellular events such asnuclear fragmentation and/or formation of apoptotic bodies bycondensation of cytoplasm, plasma membranes and/or organelles.

As used herein, the term “binding” or “specifically binding” refers tothe binding of the antibody to an epitope of the tumor antigen in an invitro assay. The in vitro assay can be a plasmon resonance assay(BIAcore, GE-Healthcare Uppsala, Sweden) with purified wild-typeantigen. For anti-CD20 antibodies the in vitro assay is preferably ascatchard analysis. The affinity of the binding is defined by the termKD. Binding or specifically binding means a binding affinity (KD) of10⁻⁸ M or less, preferably 10⁻⁸ M to 10⁻¹³ M, more preferably 10⁻⁹ M to10⁻¹³ M.

The term “CD20,” as used herein, refers to any native CD20 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length” unprocessed CD20 as well as any form of CD20that results from processing in the cell. The term also encompassesnaturally occurring variants of CD20, e.g., splice variants or allelicvariants. CD20 (also known as B-lymphocyte antigen CD20, B-lymphocytesurface antigen B1, Leu-16, Bp35, BMS, and LFS; the sequence ischaracterized by the SwissProt database entry P11836) is a hydrophobictransmembrane protein with a molecular weight of approximately 35 kDlocated on pre-B and mature B lymphocytes (Valentine, M. A. et al., J.Biol. Chem. 264 (1989) 11282-11287; Tedder, T. F., et al., Proc. Natl.Acad. Sci. U.S.A. 85 (1988) 208-212; Stamenkovic, I., et al., J. Exp.Med. 167 (1988) 1975-1980; Einfeld, D. A., et al., EMBO J. 7 (1988)711-717; Tedder, T. F., et al., J. Immunol. 142 (1989) 2560-2568). Thecorresponding human gene is Membrane-spanning 4-domains, subfamily A,member 1, also known as MS4A1. This gene encodes a member of themembrane-spanning 4A gene family. Members of this nascent protein familyare characterized by common structural features and similar intron/exonsplice boundaries and display unique expression patterns amonghematopoietic cells and nonlymphoid tissues. This gene encodes theB-lymphocyte surface molecule which plays a role in the development anddifferentiation of B-cells into plasma cells. This family member islocalized to 11q12, among a cluster of family members. Alternativesplicing of this gene results in two transcript variants which encodethe same protein. The terms “CD20” and “CD20 antigen” are usedinterchangeably herein, and include any variants, isoforms and specieshomologs of human CD20 which are naturally expressed by cells or areexpressed on cells transfected with the CD20 gene. Binding of anantibody of the invention to the CD20 antigen mediate the killing ofcells expressing CD20 by signaling through CD20, by inactivating CD20 orby cross-linking CD20. Preferably, the cell is a tumor cell. The killingof the cells expressing CD20 may occur by one or more of the followingmechanisms: Direct cell death/apoptosis induction, ADCC, CDC and ADCP.Synonyms of CD20, as recognized in the art, include B-lymphocyte antigenCD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BMS, and LFS.

The term “CD20 expressing cancer” as used herein refers preferably tolymphomas (preferably B-Cell Non-Hodgkin's lymphomas (NHL)) andlymphocytic leukemias. Such lymphomas and lymphocytic leukemias includefollicular lymphomas, Small Non-Cleaved Cell Lymphomas/Burkitt'slymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt'slymphoma and Non-Burkitt's lymphoma), marginal zone lymphomas (includingextranodal marginal zone B cell lymphoma (Mucosa-associated lymphatictissue lymphomas, MALT), nodal marginal zone B cell lymphoma and splenicmarginal zone lymphoma), Mantle cell lymphoma (MCL), Large Cell Lymphoma(including diffuse large B-cell lymphoma (DLBCL), Diffuse Mixed CellLymphoma, Immunoblastic Lymphoma, Primary Mediastinal B-Cell Lymphoma,Angiocentric Lymphoma-Pulmonary B-Cell Lymphoma), hairy cell leukemia,lymphocytic lymphoma, Waldenstrom's macroglobulinemia, acute lymphocyticleukemia (ALL), chronic lymphocytic leukemia (CLL)/small lymphocyticlymphoma (SLL), B-cell prolymphocytic leukemia, plasma cell neoplasms,plasma cell myeloma, multiple myeloma, plasmacytoma, Hodgkin's disease.More preferably, the term CD20 expressing cancer refers to Non-Hodgkin'slymphomas (NHL), follicular lymphomas, diffuse large B-cell lymphoma(DLBCL) and chronic lymphocytic leukemia (CLL).

The term “complement-dependent cytotoxicity (CDC)” refers to lysis ofhuman tumor target cells by the antibody according to the invention inthe presence of complement. CDC is measured preferably by the treatmentof a preparation of CD20 expressing cells with an anti-CD20 antibodyaccording to the invention in the presence of complement. CDC is foundif the antibody induces at a concentration of 100 nM the lysis (celldeath) of 20% or more of the tumor cells after 4 hours. The assay isperformed preferably with ⁵¹Cr or Eu labeled tumor cells and measurementof released ⁵¹Cr or Eu. Controls include the incubation of the tumortarget cells with complement but without the antibody.

Typically, type I and type II anti-CD20 antibodies of the IgG1 isotypeshow characteristic CDC properties. Type I anti-CD20 antibodies have anincreased CDC (if IgG1 isotype) and type II anti-CD20 antibodies have adecreased CDC (if IgG1 isotype) compared to each other. Preferably bothtype I and type II anti-CD20 antibodies are IgG1 isotype antibodies.

As used herein, “cell signaling mechanism” or “cell signaling activity”is intended to refer to the entire signaling (i.e., signal transduction)pathway that leads to a particular cellular event or biologicalfunction, as well as any signaling steps along the pathway.

As used herein, the term “CH1 region” is intended to refer to the domainof the heavy chain of an immunoglobulin that is just C-terminal to thevariable region and N-terminal to the hinge region. In an immunoglobulinof the IgG type, for example, CH1 is normally defined by Kabat positions114 to 223 (Kabat numbering).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarily determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991) and by Chothia et al., J MoI Biol. 196:901-917(1987), each of which is hereby incorporated by reference in itsentirety, where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The exact residue numbers which encompass aparticular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine which residues comprisea particular CDR given the variable region amino acid sequence of theantibody.

“Conservative” amino acid substitutions are those made by replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements, and maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature, and/orthe bulk sizes of the residues involved. For example, nonpolar(hydrophobic) amino acids include glycine, alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan, and methionine; polarneutral amino acids include serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. “Substitutions”,“insertions” or “deletions” are preferably in the range of about 1 to 20amino acids, more preferably 1 to 10 amino acids, more preferably 1 to 4amino acids, most preferably 1 amino acid. The variation allowed may beexperimentally determined by systematically making insertions,deletions, or substitutions of amino acids in a polypeptide moleculeusing recombinant DNA techniques and assaying the resulting recombinantvariants for activity.

“Cytokine release syndrome”, which is an “infusion reaction”, is acommon immediate complication occurring with the use of antibodyinfusions such as e.g., the CD20-antibody rituximab. The pathogenesis ischaracterized in that the antibodies bind to T cell receptors,activating said T cells. The cytokines released by the activated T cellsproduce a type of systemic inflammatory response similar to that foundin severe infection characterised by hypotension, pyrexia and rigors.Deaths due to cytokine release syndrome have been reported, and it cancause life-threatening pulmonary edema if the patient is fluidoverloaded.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (including but not limited to, ²¹¹Astatine,¹³¹Iodine, ¹²⁵Iodine, ⁹⁰Yttrium, ¹⁸⁶Rhenium, ¹⁸⁸Rhenium, ¹⁵³Samarium,²¹²Bismuth, ³²Phosphorus, ²¹²Lead and radioactive isotopes of Lutetium);chemotherapeutic agents or drugs (including but not limited to,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents); growth inhibitory agents;enzymes and fragments thereof such as nucleolytic enzymes; antibiotics;toxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof; and the various antitumor or anticancer agentsdisclosed below.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

As used herein, the term “effector function” or “Fc-mediated effectorfunction” refers to those biological activities attributable to the Fcregion (a native sequence Fc region or amino acid sequence variant Fcregion) of an antibody, and vary with the antibody isotype. Examples ofantibody effector functions include, but are not limited to: C1q bindingand complement dependent cytotoxicity (CDC), Fc receptor bindingaffinity, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion.As used herein, the term “reduced” in conjunction with effector functionrefers to a measurable reduction of effector function induced by anantibody modified according to the invention compared to effectorfunction induced by the corresponding parent non-substituted antibody.Effector function can be measured as disclosed herein and with referenceto the examples as disclosed herein. As used herein, the terms “stronglyreduced”, “abolished” and “residual” in conjunction with effectorfunction are considered to refer to a reduction of the named effectorfunction induced by an antibody modified according to the inventioncompared to effector function induced by the corresponding parentnon-substituted antibody. “Strongly reduced” means a reduction to 50% orless, “abolished” means a reduction to 10% or less and “residual” meansmore than 10% compared to the respective effector function induced bythe corresponding parent non-substituted antibody. Accordingly,antibodies of the present invention comprising an Fc variant of theinvention comprise at least one or more of the following properties:reduced or abolished ADCC, reduced or abolished CDC, reduced orabolished ADCP, reduced or abolished binding to Fc receptors, reduced orabolished binding to C1q and reduced or abolished infusion reaction(cytokine release syndrome). In one preferred embodiment, provided is anantibody wherein ADCP function induced by the antibody comprising thevariant heavy chain region is reduced compared to ADCP function inducedby the parent non-substituted antibody, wherein more than 10% of ADCPfunction induced by the antibody comprising the variant heavy chainregion is retained compared to ADCP function induced by the parentnon-substituted antibody.

As used herein, the terms “engineer, engineered, engineering,glycoengineer, glycoengineered, glycoengineering”, “glycosylationengineering” and “GE” are considered to include any manipulation of theglycosylation pattern of a naturally occurring or recombinantpolypeptide, such as an antibody, or fragment thereof. Glycosylationengineering includes metabolic engineering of the glycosylationmachinery of a cell, including genetic manipulations of theoligosaccharide synthesis pathways to achieve altered glycosylation ofglycoproteins expressed in cells. In one embodiment, the glycosylationengineering is an alteration in glycosyltransferase activity. In aparticular embodiment, the engineering results in alteredglucosaminyltransferase activity and/or fucosyltransferase activity.

The term “expression of the CD20 antigen” is intended to indicate asignificant level of expression of the CD20 antigen in a cell,preferably on the cell surface of a T- or B-cell, more preferably aB-cell, from a tumor or cancer, respectively, preferably a non-solidtumor. Patients having a “CD20 expressing cancer” can be determined bystandard assays known in the art. “Expression of the CD20” antigen isalso preferable intended to indicate a significant level of expressionof the CD20 antigen in a cell, preferably on the cell surface of a T- orB-cell, more preferably a B-cell, in an autoimmune disease. CD20 antigenexpression is measured e.g., using immunohistochemical (IHC) detection,FACS or via PCR-based detection of the corresponding mRNA.

As used herein, the term “Fc region” is intended to refer to aC-terminal region of an IgG heavy chain. Although the boundaries of theFc region of an IgG heavy chain might vary slightly, the human IgG heavychain Fc region is usually defined to stretch from the amino acidresidue at position Cys226 to the carboxyl-terminus.

As used herein, the term “Fc-mediated cellular cytotoxicity” includes“antibody-dependent cell-mediated cytotoxicity” (ADCC) and cellularcytotoxicity mediated by a soluble Fc-fusion protein containing a humanFc-region. It is an immune mechanism leading to the lysis of“antibody-targeted cells” by “human immune effector cells”, wherein thehuman immune effector cells are a population of leukocytes that displayFc receptors on their surface through which they bind to the Fc-regionof antibodies or of Fc-fusion proteins and perform effector functions.Such a population may include, but is not limited to, peripheral bloodmononuclear cells (PBMC) and/or natural killer (NK) cells. Theantibody-targeted cells are cells bound by the antibodies or Fc-fusionproteins. The antibodies or Fc fusion-proteins bind to target cells viathe protein part N-terminal to the Fc region.

As used herein, the terms “fusion” and “chimeric”, when used inreference to polypeptides such as antibodies refer to polypeptidescomprising amino acid sequences derived from two or more heterologouspolypeptides, such as portions of antibodies from different species. Forchimeric antibodies, for example, the non-antigen binding components maybe derived from a wide variety of species, including primates such aschimpanzees and humans. The constant region of the chimeric antibody ismost preferably substantially identical to the constant region of anatural human antibody; the variable region of the chimeric antibody ismost preferably derived from a non-human (i.e., donor) antigen bindingmolecule that specifically binds an antigen of interest. The chimericantibody may comprise the entire donor variable region; alternatively,the chimeric antibody may comprise a humanized or primatized antibody.Humanized antibodies are a particularly preferred form of fusion orchimeric antibody. Other forms of “chimeric antibodies” encompassed bythe present invention are those in which the class or subclass has beenmodified or changed from that of the original antibody. Such “chimeric”antibodies are also referred to as “class-switched antibodies”. Methodsfor producing chimeric antibodies involve conventional recombinant DNAand gene transfection techniques now well known in the art. See, e.g.,Morrison, S. L., et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855;U.S. Pat. Nos. 5,202,238 and 5,204,244.

As used herein, the term “heavy chain variable region” is intended torefer to the N-terminal domain of an immunoglobulin heavy chain. In oneexample, the heavy chain variable region is defined by Kabat positions 1to 113 (with possible insertions at particular residues as designated byKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)). According to one embodiment of the present invention, amodified antibody can comprise a functional fragment of a heavy chainvariable region.

As used herein, the term “heavy chain constant region” is intended torefer to the C terminal domain of an immunoglobulin heavy chain. Thereare five naturally-occurring classes of heavy chain constant regions:IgA, IgG, IgE, IgD, and IgM. In one example, the heavy chain constantregion comprises a CH1 domain, a CH2 domain, and a CH3 domain.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). Disclosed also in van Dijk and van deWinkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies canbe prepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (disclosed, e.g. in, U.S. Pat. Nos. 6,075,181 and 6,150,584regarding XENOMOUSE™ technology). See also, for example, Li et al.,Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding humanantibodies generated via a human B-cell hybridoma technology.

As used herein, the term “humanized” is used to refer to an antigenbinding molecule derived from a non-human antigen-binding molecule, forexample, a murine antibody, that retains or substantially retains theantigen-binding properties of the parent molecule but which is lessimmunogenic in humans. This may be achieved by various methods including(a) grafting the entire non-human variable domains onto human constantregions to generate chimeric antibodies, (b) grafting only the non-humanCDRs onto human framework and constant regions with or without retentionof critical framework residues (e.g., those that are important forretaining good antigen binding affinity or antibody functions), or (c)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like section by replacement of surface residues. Suchmethods are disclosed in Jones et al., Morrison et al., Proc. Natl.Acad. Sd., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol, 44:65-92(1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec.Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994),each of which is hereby incorporated by reference in its entirety. Thereare generally three complementarity determining regions (CDRs) (CDR1,CDR2 and CDR3), in each of the heavy and light chain variable domains ofan antibody, which are flanked by four framework subregions (i.e., FR1,FR2, FR3, and FR4) in each of the heavy and light chain variable domainsof an antibody: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A discussion ofhumanized antibodies can be found, inter alia, in U.S. Pat. No.6,632,927, and in published U.S. Application No. 2003/0175269, each ofwhich is hereby incorporated by reference in its entirety. Similarly, asused herein, the term “primatized” is used to refer to anantigen-binding molecule derived from a non-primate antigen-bindingmolecule, for example, a murine antibody, that retains or substantiallyretains the antigen-binding properties of the parent molecule but whichis less immunogenic in primates.

As used herein, a nucleic acid that “hybridizes under stringentconditions” to a nucleic acid sequence of the invention, refers to apolynucleotide that hybridizes in an overnight incubation at 42° C. in asolution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C.

As used herein, the term “host cell” covers any kind of cellular systemwhich can be engineered to generate the polypeptides and antigen-bindingmolecules of the present invention. Host cells include cultured cells,including but not limited to, mammalian cultured cells, such as CHOcells, BHK cells, HEK293-EBNA cells, NSO cells, SP2/0 cells, Y0 myelomacells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridomacells, yeast cells, insect cells, and plant cells, to name only a few,but also cells comprised within a transgenic animal, transgenic plant orcultured plant or animal tissue. In one embodiment, the host cell isengineered to allow the production of an antigen binding molecule withmodified glycoforms. In a preferred embodiment, the antigen bindingmolecule is an antibody, antibody fragment, or fusion protein.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated antibody” is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). A review of methods for assessment of antibody purity isdisclosed, inter alia, in Flatman et al., J. Chromatogr. B 848:79-87(2007).

An “isolated nucleic acid” refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-CD20 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being disclosed herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150′000 daltons, composed oftwo identical “light chains” and two identical “heavy chains” that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein, the term “parent antibody”, or “parent non-modifiedantibody”, or “parent non-substituted antibody” refers to an antibodyhaving a particular amino acid sequence encoded by a polynucleotidesequence. The sequence of the parent molecule (i.e., the “parentsequence”) serves as a reference sequence for making amino acid residuesubstitutions that alter the ability of the resulting molecule to induceeffector function and/or to induce signaling activity and/orcross-linking of antigen. Likewise, the activity of a parent molecule(e.g., the “parent non-substituted antibody) serves as the referencewhen determining whether a substitution has an effect on effectorfunction and/or cell signaling activity and/or cross-linking of antigen,and, where relevant, the extent of that effect.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, comprising natural or non-naturalamino acid residues, and are not limited to a minimum length. Thus,peptides, oligopeptides, dimers, multimers, and the like are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also includepost-translational modifications of the polypeptide, including, forexample, glycosylation, sialylation, acetylation, and phosphorylation.

Furthermore, a “polypeptide” herein also refers to a modified proteinsuch as single or multiple amino acid residue deletions, additions, andsubstitutions to the native sequence, as long as the protein maintains adesired activity. For example, a serine residue may be substituted toeliminate a single reactive cysteine or to remove disulfide bonding or aconservative amino acid substitution may be made to eliminate a cleavagesite. These modifications may be deliberate, as through site-directedmutagenesis, or may be accidental, such as through mutations of hostswhich produce the proteins or errors due to polymerase chain reaction(PCR) amplification.

The “ratio of the binding capacities to CD20 on Raji cells (ATCC-No.CCL-86) of an anti-CD20 antibodies compared to rituximab” is determinedby direct immunofluorescence measurement (the mean fluorescentintensities (MFI) is measured) using said anti-CD20 antibody conjugatedwith Cy5 and rituximab conjugated with Cy5 in a FACSArray (BectonDickinson) with Raji cells (ATCC-No. CCL-86), and calculated as follows:

${{Ratio}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {binding}\mspace{14mu} {capacities}\mspace{14mu} {to}\mspace{14mu} {Cd}\; 20\mspace{14mu} {on}\mspace{14mu} {Raji}\mspace{14mu} {cells}\mspace{14mu} \left( {{ATCC}\text{-}{{No}.\mspace{14mu} {CCL}}\text{-}86} \right)} = {\frac{{MFI}\left( {{Cy}\; 5\text{-}{anti}\text{-}{CD}\; 20\mspace{14mu} {antibody}} \right)}{{MFI}\left( {{Cy}\; 5\text{-}{rituximab}} \right)} \times \frac{{Cy}\; 5\text{-}{labeling}\mspace{14mu} {ratio}\mspace{14mu} \left( {{Cy}\; 5\text{-}{rituximab}} \right)}{{Cy}\; 5\text{-}{labeling}\mspace{14mu} {ratio}\mspace{14mu} \left( {{Cy}\; 5\text{-}{anti}\text{-}{CD}\; 20\mspace{14mu} {antibody}} \right)}}$

MFI is the mean fluorescent intensity. The “Cy5-labeling ratio” as usedherein means number of Cy5-label molecules per molecule antibody.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from a hostcell such as a NSO or CHO cell or from an animal (e.g. a mouse) that istransgenic for human immunoglobulin genes or antibodies expressed usinga recombinant expression vector transfected into a host cell. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences in a rearranged form. Therecombinant human antibodies according to the invention have beensubjected to in vivo somatic hypermutation. Thus, the amino acidsequences of the recombinant antibodies are sequences that, whilederived from and related to human germline sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH”. Thevariable domain of the light chain may be referred to as “VL”. Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

By “variant protein”, “protein variant” or “variant” as used herein ismeant a protein that differs from that of a parent protein by virtue ofat least one amino acid modification. Variant refers to the proteinitself, a composition comprising the polypeptide, or the amino sequencethat encodes it. Preferably, the protein variant has at least one aminoacid modification compared to the parent protein, e.g. from about one toabout seventy amino acid modifications, and preferably from about one toabout five amino acid modifications compared to the parent. The proteinvariant sequence as disclosed herein will preferably possess at leastabout 80% homology with a parent protein sequence, and most preferablyat least about 90% homology, more preferably at least about 95%homology. Variant protein can refer to the variant protein itself,compositions comprising the protein variant, or the DNA sequence thatencodes it. Accordingly, by “antibody variant”, “variant antibody” or“variant heavy chain region” as used herein is meant an antibody, orpart of an antibody, that differs from a parent antibody by virtue of atleast one amino acid modification including but not limited to aminoacid substitution, deletion or insertion. By “IgG variant” or “variantIgG” as used herein is meant an antibody that differs from a parent IgGby virtue of at least one amino acid modification, and “immunoglobulinvariant” or “variant immunoglobulin” as used herein is meant animmunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification.

In the context of the present invention, the term “variant” is usedinterchangeably with the term “mutated”. Accordingly, by “antibodymutant”, “mutated antibody” or “mutated heavy chain region” as usedherein is meant an antibody, or part of an antibody, that differs from aparent antibody by virtue of at least one amino acid modificationincluding but not limited to amino acid substitution, deletion orinsertion. “IgG mutant” or “mutated IgG” as used herein is meant anantibody that differs from a parent IgG by virtue of at least one aminoacid modification, and “immunoglobulin mutant” or “mutantimmunoglobulin” as used herein is meant an immunoglobulin sequence thatdiffers from that of a parent immunoglobulin sequence by virtue of atleast one amino acid modification.

The term “variable” in relation with the term “variable domain” refersto the fact that certain segments of the variable domains differextensively in sequence among antibodies. The V domain mediates antigenbinding and defines specificity of a particular antibody for itsparticular antigen. However, the variability is not evenly distributedacross the 110-amino acid span of the variable domains. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-12amino acids long. The variable domains of native heavy and light chainseach comprise four FRs, largely adopting a β-sheet configuration,connected by three hypervariable regions, which form loops connecting,and in some cases forming part of, the β-sheet structure. Thehypervariable regions in each chain are held together in close proximityby the FRs and, with the hypervariable regions from the other chain,contribute to the formation of the antigen-binding site of antibodies(see Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)). The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cell-mediatedcytotoxicity (ADCC).

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. Recombinantvariants encoding same or similar polypeptides may be synthesized orselected by making use of the “redundancy” in the genetic code. Variouscodon substitutions, such as the silent changes which produce variousrestriction sites, may be introduced to optimize cloning into a plasmidor viral vector or expression in a particular prokaryotic or eukaryoticsystem. Mutations in the polynucleotide sequence may be reflected in thepolypeptide or domains of other peptides added to the polypeptide tomodify the properties of any part of the polypeptide, to changecharacteristics such as ligand-binding affinities, interchainaffinities, or degradation/turnover rate.

The term “wildtype polypeptide” and “wildtype (human) Fc region” as usedherein refers to a polypeptide and Fc region, respectively, comprisingan amino acid sequence which lacks one or more of the Fc regionmodifications disclosed herein, because they have not been introduced,and serve for example as controls. The wildtype polypeptide may comprisea native sequence Fc region or an Fc region with pre-existing amino acidsequence modifications (such as additions, deletions and/orsubstitutions).

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence or polypeptide sequence of thepresent invention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al, Comp. App. Biosci. 6:237-245 (1990). In a sequencealignment the query and subject sequences are both DNA sequences. An RNAsequence can be compared by converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5,Gap Size Penalty 0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the FASTDB program as disclosed herein using the specifiedparameters, to arrive at a final percent identity score. This correctedscore is what is used for the purposes of the present invention. Onlybases outside the 5′ and 3′ bases of the subject sequence, as displayedby the FASTDB alignment, which are not matched/aligned with the querysequence, are calculated for the purposes of manually adjusting thepercent identity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at the 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ end of the subject sequence which are not matched/aligned withthe query. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases on the 5′ and 3′ end of thesubject sequence which are not matched/aligned with the query sequenceare manually corrected for. No other manual corrections are to be madefor the purposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a referencepolypeptide can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al, Comp. App. Biosci. 6:237-245 (1990). In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=O, CutoffScore=1, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or thelength of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the FASTDB program as disclosed herein using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thepresent invention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to be made forthe purposes of the present invention.

II. Embodiments According to the Invention Modified Antibodies Accordingto the Invention

There remains a need for monoclonal antibodies with improved therapeuticpotential for human therapy. Enforcing the induction of direct celldeath while reducing effector function, is much needed for improvedcancer therapy. Unexpectedly, the present inventors found that thesubstitutions Asn297Asp or Pro329Gly in the Fc region as disclosedherein strongly reduced CDC and ADCC function but retained residual ADCPfunction. Further surprisingly, this mutation can be combined withmutations in the elbow hinge region according to the present invention,leading to altered (increased or reduced) direct cell death induction.In combination, the modifications according to the present inventionallow a selective and independent modulation of induction of effectorfunction and/or induction of direct cell death as compared tonon-substituted parent antibodies.

The present inventors surprisingly found that substitution of theasparagine residue at Kabat position 297 or of the proline residue atKabat position 329 leads to reduced effector function. Morespecifically, substitution of the asparagine residue at position 297 toaspartic acid or of the proline residue at Kabat position 329 to glycineresulted with strongly reduced or abolished ADCC and CDC effectorfunctions but residual ADCP function. Accordingly, in one embodiment, anantibody is provided, comprising a variant heavy chain region comprisingat least one amino acid substitution relative to the parentnon-substituted heavy chain region, wherein the heavy chain region ofthe parent non-substituted antibody comprises the amino acid residueAsn297, and wherein said substitution is Asn297Asp. In a furtherembodiment, an antibody is provided, comprising a variant heavy chainregion comprising at least one amino acid substitution relative to theparent non-substituted heavy chain region, wherein the heavy chainregion of the parent non-substituted antibody comprises the amino acidresidue Pro329, and wherein said substitution is Pro329Gly.

In a further aspect, an antibody is provided, wherein induction ofeffector function is reduced compared to effector function induced by anantibody comprising the parent non-substituted heavy chain region. Inone aspect, the present invention is related to antibodies with aminoacid modifications in the Fc region leading to strongly reduced orabolished ADCC and/or CDC function, residual ADCP function, reduced orabolished binding to Fc receptors, reduced or abolished binding to C1qand reduced or abolished toxicities.

In another embodiment said antibodies comprising a Fc variant exhibit areduced affinity to a human Fc receptor (FcγR) and/or a human complementreceptor as compared to the antibody comprising the wildtype human Fcregion. In a further embodiment the affinity to at least one of theFcγRI, FcγRII, FcγRIII is reduced, in a still further embodiment theaffinity to the FcγRI and FcγRIII is reduced, and in a still furtherembodiment the affinity to the FcγRII and FcγRIII is reduced and in astill further embodiment the affinity to the FcγRI, FcγRII and FcγRIIIis reduced, in still a further aspect of the invention the affinity tothe FcγRI receptor, FcγRIII receptor and C1q is reduced, and in still afurther aspect of the invention the affinity to the FcγRI, FcγRII,FcγRIII and C1q receptor is reduced.

The parent non-modified antibody can be any of any class (for example,but not limited to IgG, IgM, and IgE). In certain embodiments,antibodies of the invention are members of the IgG class of antibodies.In a specific embodiment, antibodies of the invention are of the IgG1,IgG2 or IgG4 subclass. In still another aspect of the invention theantibody comprising a heavy chain constant region variant and an Fcvariant comprises a human IgG1 or IgG4 Fc region. In still a furtheraspect of the invention the variants are IgG1 or IgG4 antibodies. In aspecific embodiment, antibodies of the invention are of the IgG1subclass, wherein the heavy chain region of the parent non-substitutedantibody comprises the amino acid residue Asn297, wherein the residuesare numbered according to the EU index as in Kabat. In a furtherspecific embodiment, antibodies of the invention are of the IgG1subclass, wherein the heavy chain region of the parent non-substitutedantibody comprises the amino acid residue Pro329, wherein the residuesare numbered according to the EU index as in Kabat. In another specificembodiment, antibodies of the invention are of the IgG1 subclass,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residue Asn297, wherein the variant heavy chainregion comprises the amino acid substitution Asn297Asp of the Fc region.In another specific embodiment, antibodies of the invention are of theIgG1 subclass, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residue Pro329,wherein the variant heavy chain region comprises the amino acidsubstitution Pro329Gly of the Fc region. In one embodiment, antibodiesof the invention comprising the amino acid substitution Pro329Gly have ahigher melting temperature compared to a variant of the parentnon-substituted antibody comprising an amino acid substitution at aminoacid residue Asn297 as measured by Tryptophan fluorescence. In oneembodiment, an antibody of the invention comprising the amino acidsubstitution Pro329Gly has a higher melting temperature compared to avariant of the parent non-substituted antibody comprising the Asn297Aspsubstitution as measured by Tryptophan fluorescence.

Parent non-substituted antibodies according to the invention include,but are not limited to, monoclonal antibodies. In one aspect antibodiesof the invention are so-called chimaeric antibodies, humanizedantibodies or fully human antibodies. In a further aspect antibodies ofthe invention are full length antibodies or antibody fragments havingthe same biological activity including amino acid sequence variantsand/or glycosylation variants of such antibodies or fragments. Parentnon-substituted antibodies according to the invention include, but arenot limited to, 3F8 (anti-GD2), Abagovomab (anti CA-125), Abciximab(anti CD41 (integrin alpha-IIb)), Adalimumab (anti-TNF-α), Adecatumumab(anti-EpCAM, CD326), Afelimomab (anti-TNF-α); Afutuzumab (anti-CD20),Alacizumab pegol (anti-VEGFR2), ALD518 (anti-IL-6), Alemtuzumab(Campath, MabCampath, anti-CD52), Altumomab (anti-CEA), Anatumomab(anti-TAG-72), Anrukinzumab (IMA-638, anti-IL-13), Apolizumab(anti-HLA-DR), Arcitumomab (anti-CEA), Aselizumab (anti-L-selectin,CD62L), Atlizumab (tocilizumab, Actemra, RoActemra, anti-IL-6 receptor),Atorolimumab (anti-Rhesus factor), Bapineuzumab (anti-beta amyloid),Basiliximab (Simulect, antiCD25 (α chain of IL-2 receptor)), Bavituximab(anti-phosphatidylserine), Bectumomab (LymphoScan, anti-CD22), Belimumab(Benlysta, LymphoStat-B, anti-BAFF), Benralizumab (anti-CD125),Bertilimumab (anti-CCL11 (eotaxin-1)), Besilesomab (Scintimun,anti-CEA-related antigen), Bevacizumab (Avastin, anti-VEGF-A), Biciromab(FibriScint, anti-fibrin II beta chain), Bivatuzumab (anti-CD44 v6),Blinatumomab (BiTE, anti-CD19), Brentuximab (cAC10, anti-CD30 TNFRSF8),Briakinumab (anti-IL-12, IL23), Canakinumab (Ilaris, anti-IL-1),Cantuzumab (C242, anti-CanAg), Capromab, Catumaxomab (Removab,anti-EpCAM, anti-CD3), CC49 (anti-TAG-72), Cedelizumab (anti-CD4),Certolizumab pegol (Cimzia anti-TNF-α), Cetuximab (Erbitux, IMC-C225,anti-EGFR), Citatuzumab bogatox (anti-EpCAM), Cixutumumab (anti-IGF-1),Clenoliximab (anti-CD4), Clivatuzumab (anti-MUC 1), Conatumumab(anti-TRAIL-R2), CR6261 (anti-Influenza A hemagglutinin), Dacetuzumab(anti-CD40), Daclizumab (Zenapax, anti-CD25 (a chain of IL-2 receptor)),Daratumumab (anti-CD38, cyclic ADP ribose hydrolase), Denosumab (Prolia,anti-RANKL), Detumomab (anti-B-lymphoma cell), Dorlimomab, Dorlixizumab,Ecromeximab (anti-GD3 ganglioside), Eculizumab (Soliris, anti-C5),Edobacomab (anti-endotoxin), Edrecolomab (Panorex, MAb17-1A,anti-EpCAM), Efalizumab (Raptiva, anti-LFA-1 (CD 11a)), Efungumab(Mycograb, anti-Hsp90), Elotuzumab (anti-SLAMF7), Elsilimomab(anti-IL-6), Enlimomab pegol (anti-ICAM-1 (CD54)), Epitumomab(anti-episialin), Epratuzumab (anti-CD22), Erlizumab (anti-ITGB2 (CD18)), Ertumaxomab (Rexomun, anti-HER2/neu, CD3), Etaracizumab (Abegrin,anti-integrin α_(v)β₃), Exbivirumab (anti-hepatitis B surface antigen),Fanolesomab (NeutroSpec, anti-CD 15), Faralimomab (anti-interferonreceptor), Farletuzumab (anti-folate receptor 1), Felvizumab(anti-respiratory syncytial virus), Fezakinumab (anti-IL-22),Figitumumab (anti-IGF-1 receptor), Fontolizumab (anti-IFN-γ),Foravirumab (anti-rabies virus glycoprotein), Fresolimumab (anti-TGF-β),Galiximab (anti-CD80), Gantenerumab (anti-beta amyloid), Gavilimomab(anti-CD147 (basigin)), Gemtuzumab (anti-CD33), Girentuximab(anti-carbonic anhydrase 9), Glembatumumab (CR011, anti-GPNMB),Golimumab (Simponi, anti-TNF-a), Gomiliximab (anti-CD23 (IgE receptor)),Ibalizumab (anti-CD4), Ibritumomab (anti-CD20), Igovomab (Indimacis-125,anti-CA-125), Imciromab (Myoscint, anti-cardiac myosin), Infliximab(Remicade, anti-TNF-a), Intetumumab (anti-CD51), Inolimomab (anti-CD25(a chain of IL-2 receptor)), Inotuzumab (anti-CD22), Ipilimumab (anti-CD152), Iratumumab (anti-CD30 (TNFRSF8)), Keliximab (anti-CD4),Labetuzumab (CEA-Cide, anti-CEA), Lebrikizumab (anti-IL-13). Lemalesomab(anti-NCA-90 (granulocyte antigen)), Lerdelimumab (anti-TGF beta 2),Lexatumumab (anti-TRAIL-R2), Libivirumab (anti-hepatitis B surfaceantigen), Lintuzumab (anti-CD33)), Lucatumumab (anti-CD40), Lumiliximab(anti-CD23 (IgE receptor), Mapatumumab (anti-TRAIL-R1), Maslimomab(anti-T-cell receptor), Matuzumab (anti-EGFR), Mepolizumab (Bosatria,anti-IL-5), Metelimumab (anti-TGF beta 1), Milatuzumab (anti-CD74),Minretumomab (anti-TAG-72), Mitumomab (BEC-2, anti-GD3 ganglioside),Morolimumab (anti-Rhesus factor), Motavizumab (Numax, anti-respiratorysyncytial virus), Muromonab-CD3 (Orthoclone OKT3, anti-CD3), Nacolomab(anti-C242), Naptumomab (anti-5T4), Natalizumab (Tysabri, anti-integrinα4), Nebacumab (anti-endotoxin), Necitumumab (anti-EGFR), Nerelimomab(anti-TNF-a), Nimotuzumab (Theracim, Theraloc, anti-EGFR), Nofetumomab,Obinutuzumab (anti-CD20), Ocrelizumab (anti-CD20), Odulimomab(Afolimomab, anti-LFA-1 (CD11a)), Ofatumumab (Arzerra, anti-CD20),Olaratumab (anti-PDGF-Ra), Omalizumab (Xolair, anti-IgE Fc region),Oportuzumab (anti-EpCAM), Oregovomab (OvaRex, anti-CA-125), Otelixizumab(anti-CD3), Pagibaximab (anti-lipoteichoic acid), Palivizumab (Synagis,Abbosynagis, anti-respiratory syncytial virus), Panitumumab (Vectibix,ABX-EGF, anti-EGFR), Panobacumab (anti-Pseudomonas aeruginosa),Pascolizumab (anti-IL-4), Pemtumomab (Theragyn, anti-MUC1), Pertuzumab(Omnitarg, 2C4, anti-IIER2/neu), Pexelizumab (anti-05), Pintumomab(anti-adenocarcinoma antigen), Priliximab (anti-CD4), Pritumumab(anti-vimentin), PRO 140 (anti-CCRS), Racotumomab (1E10,anti-(N-glycolylneuraminic acid (NeuGc, NGNA)-gangliosides GM3)),Rafivirumab (anti-rabies virus glycoprotein), Ramucirumab (anti-VEGFR2),Ranibizumab (Lucentis, anti-VEGF-A), Raxibacumab (anti-anthrax toxin,protective antigen), Regavirumab (anti-cytomegalovirus glycoprotein B),Reslizumab (anti-IL-5), Rilotumumab (anti-HGF), Rituximab (MabThera,Rituxan, anti-CD20), Robatumumab (anti-IGF-1 receptor), Rontalizumab(anti-IFN-a), Rovelizumab (LeukArrest, anti-CD11, CD 18), Ruplizumab(Antova, anti-CD 154 (CD40L)), Satumomab (anti-TAG-72), Sevirumab(anti-cytomegalovirus), Sibrotuzumab (anti-FAP), Sifalimumab(anti-IFN-a), Sltuximab (anti-IL-6), Siplizumab (anti-CD2), (Smart) MI95(anti-CD33), Solanezumab (anti-beta amyloid), Sonepcizumab(anti-spingosine-1-phosphate), Sontuzumab (anti-episialin), Stamulumab(anti-myostatin), Sulesomab (LeukoScan, (anti-NCA-90 (granulocyteantigen), Tacatuzumab (anti-alpha-fetoprotein), Tadocizumab(anti-integrin α_(IIb)β₃), Talizumab (anti-IgE), Tanezumab (anti-NGF),Taplitumomab (anti-CD19), Tefibazumab (Aurexis, (anti-clumping factorA)), Telimomab, Tenatumomab (anti-tenascin C), Teneliximab (anti-CD40),Teplizumab (anti-CD3), TGN1412 (anti-CD28), Ticilimumab (Tremelimumab,(anti-CTLA-4)), Tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13),Tocilizumab (Atlizumab, Actemra, RoActemra, (anti-IL-6 receptor)),Toralizumab (anti-CD 154 (CD40L)), Tositumomab (anti-CD20), Trastuzumab(Herceptin, (anti-HER2/neu)), Tremelimumab (anti-CTLA-4), Tucotuzumabcelmoleukin (anti-EpCAM), Tuvirumab (anti-hepatitis B virus),Urtoxazumab (anti-Escherichia coli), Ustekinumab (Stelara, anti-IL-12,IL-23), Vapaliximab (anti-AOC3 (VAP-1)), Vedolizumab (anti-integrinα₄β₇), Veltuzumab (anti-CD20), Vepalimomab (anti-AOC3 (VAP-1),Visilizumab (Nuvion, anti-CD3), Vitaxin (anti-vascular integrin avb3),Volociximab (anti-integrin α₅β₁), Votumumab (HumaSPECT, anti-tumorantigen CTAA16.88), Zalutumumab (HuMax-EGFr, (anti-EGFR)), Zanolimumab(HuMax-CD4, anti-CD4), Ziralimumab (anti-CD 147 (basigin)), Zolimomab(anti-CD5), Etanercept (Enbrel®), Alefacept (Amevive®), Abatacept(Orencia®), Rilonacept (Arcalyst), 14F7 (anti-IRP-2 (Iron RegulatoryProtein 2)), 14G2a (anti-GD2 ganglioside, from Nat. Cancer Inst, formelanoma and solid tumors), J591 (anti-PSMA, Weill Cornell MedicalSchool for prostate cancers), 225.28S (anti-HMW-MAA (High molecularweight-melanoma-antigen), Sorin Radiofarrnaci S.R.L. (Milan, Italy) formelanoma), COL-1 (anti-CEACAM3, CGM1, from Nat. Cancer Inst. USA forcolorectal and gastric cancers), CYT-356 (Oncoltad®, for prostatecancers), HNK20 (OraVax Inc. for respiratory syncytial virus), ImmuRAIT(from Immunomedics for NHL), Lym-1 (anti-HLA-DR10, Peregrine Pharm. forCancers), MAK-195F (anti-TNF (tumor necrosis factor; TNFA, TNF-alpha;TNFSF2), from Abbott/Knoll for Sepsis toxic shock), MEDI-500 (T10B9,anti-CD3, TRαβ (T cell receptor alpha/beta), complex, from Medlmmune Incfor Graft-versus-host disease), RING SCAN (anti-TAG 72 (tumourassociated glycoprotein 72), from Neoprobe Corp. for Breast, Colon andRectal cancers), Avicidin (anti-EPCAM (epithelial cell adhesionmolecule), anti-TACSTD1 (Tumor-associated calcium signal transducer 1),anti-GA733-2 (gastrointestinal tumor-associated protein 2), anti-EGP-2(epithelial glycoprotein 2); anti-KSA (KS 1/4 antigen; M4S; tumorantigen 17-1A; CD326, from NeoRx Corp. for Colon, Ovarian, Prostatecancers and NHL); LymphoCide (Immunomedics, NJ), Smart ID10 (ProteinDesign Labs), Oncolym (Techniclone Inc, CA), Allomune (BioTransplant,CA), anti-VEGF (Genentech, CA); CEAcide (Immunomedics, NJ), IMC-1C11(ImClone, NJ) and Cetuximab (ImClone, NJ).

In a preferred embodiment, the parent non-substituted antibody is ananti-CD20 antibody. Such antibodies preferably are monoclonalantibodies. Also preferably, said antibodies are selected from the groupconsisting of chimeric antibodies, humanized antibodies or fully humanantibodies. In a most preferred aspect, said antibodies are selectedfrom the group consisting of full length anti-CD20 antibodies andanti-CD20 antibody fragments having the same biological activityincluding amino acid sequence variants and glycosylation variants ofsuch antibodies or fragments. Humanized anti-CD20 parent non-substitutedantibodies according to the invention are specified with the INN namesrituximab (see e.g., U.S. Pat. No. 7,381,560 and EP2000149B1 of Andersonet. al., see e.g., FIGS. 4 and 5), ocrelizumab (as disclosed in WO2004/056312 and WO 2006/084264), ibritumomab (see WO 94/011026),tositumomab (WHO Drug Information, Vol. 12, No. 4, 1998, p. 281),veltuzumab (WHO Drug Information, Vol. 22, No. 3, 2008, p. 28) andobinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No. 4,2012, p. 453).

A parent non-substituted CD20 antibody according to the invention isrituximab (a type I anti-CD20 antibody) which is sold by Genentech Inc.and F. Hoffmann-La Roche Ltd under the trade name MABTHERA™ or RITUXAN™.Rituximab is a genetically engineered chimeric human gamma 1 murineconstant domain containing monoclonal antibody directed against thehuman CD20 antigen. This chimeric antibody contains human gamma 1constant domains and is identified by the name “C2B8” in U.S. Pat. No.5,736,137 (Anderson et. al.) issued on Apr. 17, 1998, assigned to IDECPharmaceuticals Corporation. Rituximab is approved for the treatment ofpatients with relapsed or refracting low-grade or follicular, CD20positive, B cell non-Hodgkin's lymphoma. In vitro mechanism of actionstudies have shown that rituximab exhibits human complement-dependentcytotoxicity (CDC) (Reff, M. E., et. al., Blood 83 (1994) 435-445).Additionally, it exhibits significant activity in assays that measureantibody-dependent cell-mediated cytotoxicity (ADCC). Rituximab is notafucosylated.

In a preferred embodiment the parent non-substituted antibody accordingto the invention is a humanized B-Ly1 antibody. The term “humanizedB-Ly1 antibody” refers to humanized B-Ly1 antibodies as disclosed in WO2005/044859 and WO 2007/031875, which were obtained from the murinemonoclonal anti-CD20 antibody B-Ly1 (variable region of the murine heavychain (VH): SEQ ID NO: 3; variable region of the murine light chain(VL): SEQ ID NO: 4 (see Poppema, S. and Visser, L., Biotest Bulletin 3(1987) 131-139)) by chimerization with a human constant domain from IgG1and following humanization (see WO 2005/044859 and WO 2007/031875).These humanized B Ly1 antibodies are disclosed in detail in WO2005/044859 and WO 2007/031875. In one embodiment, the humanized B-Ly1antibody has variable region of the heavy chain (VH) selected from groupof SEQ ID NO: 5 to SEQ ID NO: 21 (B-HH2 to B-HH9 and B-HL8 to B-HL17 ofWO 2005/044859 and WO 2007/031875). In one specific embodiment, suchvariable domain is selected from the group consisting of SEQ ID NOs: 5,6, 9, 11, 13, 15 and 17 (B-HH2, BHH-3, B-HH6, B-HH8, B-HL8, B-HL11 andB-HL13 of WO 2005/044859 and WO 2007/031875). In one specificembodiment, the humanized B-Ly1 antibody has a variable region of thelight chain (VL) of SEQ ID NO: 22 (B-KV1 of WO 2005/044859 and WO2007/031875). In one specific embodiment, the humanized B-Ly1 antibodyhas a variable region of the heavy chain (VH) of SEQ ID NO: 9 (B-HH6 ofWO 2005/044859 and WO 2007/031875) and a variable region of the lightchain (VL) of SEQ ID NO: 22 (B-KV1 of WO 2005/044859 and WO2007/031875). Furthermore, in one embodiment, the humanized B-Ly1antibody is an IgG1 antibody. According to one aspect of the inventionsuch afucosylated humanized B-Ly1 antibodies are glycoengineered (GE) inthe Fc region according to the procedures described in WO 2005/044859,WO 2004/065540, WO 2007/031875, Umana, P. et al., Nature Biotechnol. 17(1999) 176-180 and WO 99/154342. In one embodiment, the parentnon-substituted antibody according to the invention is the afucosylatedglycoengineered (GE) humanized B-Ly1 B-HH6-B-KV1 GE. In a preferredembodiment, the parent non-substituted antibody according to theinvention is obinutuzumab (recommended INN, WHO Drug Information, Vol.26, No. 4, 2012, p. 453). As used herein, obinutuzumab is synonymous forGA101. This replaces all previous versions (e.g. Vol. 25, No. 1, 2011,p. 75-76), and is formerly known as afutuzumab (recommended INN, WHODrug Information, Vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2, 2008, p.124).

In a preferred embodiment, the parent non-substituted antibody is a typeI anti-CD20 antibody. One essential property of type I and type IIanti-CD20 antibodies is their mode of binding. In particular, type I andtype II anti-CD20 antibodies can be classified by the ratio of thebinding capacities to CD20 on Raji cells (ATCC-No. CCL-86) of saidanti-CD20 antibody compared to rituximab. The type I anti-CD20antibodies have a ratio of the binding capacities to CD20 on Raji cells(ATCC No. CCL-86) of said anti-CD20 antibody compared to rituximab of0.8 to 1.2, preferably of 0.9 to 1.1. Preferred type I parentnon-substituted anti-CD20 antibodies include rituximab, in EP2000149B1(Anderson et. al., see FIGS. 4 and 5), 1F5 IgG2a (ECACC, hybridoma;Press et al., Blood 69/2:584-591 (1987)), HI47 IgG3 (ECACC, hybridoma),2C6 IgG1 (as disclosed in WO 2005/103081), 2F2 IgG1 or ofatumumab (asdisclosed and WO 2004/035607 and WO 2005/103081) and 2H7 IgG1 (asdisclosed in WO 2004/056312) and WO 2006/084264 (including but notlimited to the variants disclosed in tables 1 and 2). Preferably saidtype I parent non-substituted anti-CD20 antibody is a monoclonalantibody that binds to the same epitope as rituximab. In one embodiment,a type I anti-CD20 antibody is provided, comprising a variant heavychain region comprising at least one amino acid substitution relative tothe parent non-substituted heavy chain region, wherein the heavy chainregion of the parent non-substituted antibody comprises the amino acidresidue Asn297, and wherein said substitution is Asn297Asp, whereininduction of effector function is reduced compared to effector functioninduced by an antibody comprising the parent non-substituted heavy chainregion. In a preferred embodiment, the parent non-substituted antibodyis rituximab.

In another preferred embodiment, the parent non-substituted antibody isa type II anti-CD20 antibody. The type II anti-CD20 antibodies have aratio of the binding capacities to CD20 on Raji cells (ATCC No. CCL-86)of said anti-CD20 antibody compared to Rituximab of 0.3 to 0.6,preferably of 0.35 to 0.55, more preferably 0.4 to 0.5. Preferred typeII parent non-substituted anti-CD20 antibodies comprise, obinutuzumab,tositumomab (B1 IgG2a), humanized B-Ly1 antibody IgG1 (a chimerichumanized IgG1 antibody as disclosed in WO 2005/044859), 11B8 IgG1 (asdisclosed in WO 2004/035607), and AT80 IgG1. Preferably said type IIparent non-substituted anti-CD20 antibody is a monoclonal antibody thatbinds to the same epitope as humanized B-Ly1 antibody (as disclosed inWO 2005/044859). In a further embodiment, a type II anti-CD20 antibodyis provided, comprising a variant heavy chain region comprising at leastone amino acid substitution relative to the parent non-substituted heavychain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residue Asn297, andwherein said substitution is Asn297Asp, wherein induction of effectorfunction is reduced compared to effector function induced by an antibodycomprising the parent non-substituted heavy chain region. In a preferredembodiment, the parent non-substituted antibody is obinutuzumab.

The wildtype polypeptide comprises an Fc region. Generally the Fc regionof the wildtype polypeptide comprises a native or wildtype sequence Fcregion, and preferably a human native sequence Fc region (human Fcregion). However, the Fc region of the wildtype polypeptide may have oneor more pre-existing amino acid sequence alterations or modificationsfrom a native sequence Fc region. For example, the C1q or Fcγ bindingactivity of the Fc region may have been previously altered (other typesof Fc region modifications are described in more detail herein). In afurther embodiment the parent polypeptide Fc region is “conceptual” and,while it does not physically exist, the person skilled in the artselects a desired variant Fc region amino acid sequence and generates apolypeptide comprising that sequence or a DNA encoding the desiredvariant Fc region amino acid sequence. In the preferred embodiment ofthe invention, however, a nucleic acid encoding an Fc region of awildtype polypeptide (e.g., a wildtype heavy chain region) is availableand this nucleic acid sequence is altered to generate a variant nucleicacid sequence encoding the Fc region variant.

One embodiment of the invention encompasses polypeptides comprising anFc region of an antibody, comprising the addition, substitution, ordeletion of at least one amino acid residue to the Fc region resultingin reduced or abolished affinity for at least one Fc receptor. The Fcregion interacts with a number of receptors or ligands including but notlimited to Fc receptors (e.g., FcγRI, FcγRII, FcγRIII), the complementprotein C1q, and other molecules such as proteins A and G. Theseinteractions are essential for a variety of effector functions anddownstream signaling events including, but not limited to, antibodydependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP) and complement dependent cytotoxicity (CDC).Accordingly, in certain embodiments the variants of the invention havereduced or abolished affinity for an Fc receptor responsible for aneffector function compared to a polypeptide having the same amino acidsequence as the polypeptide comprising a Fc variant of the invention butnot comprising the addition, substitution, or deletion of at least oneamino acid residue to the Fc region (also referred to herein as “parentnon-substituted” polypeptide or antibody). In certain embodiments,antibodies comprising a Fc variant of the invention comprise at leastone or more of the following properties: reduced or abolished effector(ADCC and/or CDC and/or ADCP) function, reduced or abolished binding toFc receptors, reduced or abolished binding to C1q and reduced orabolished infusion reaction (cytokine release syndrome). Morespecifically, embodiments of the invention provide anti-CD20 (same asobinutuzumab or rituximab), anti-CD9 (same as TA), anti-Selectin (pSel),anti-CD37, anti-HER2 and anti-EGFR antibodies with reduced affinity forFc receptors (e.g. FcγRI, FcγRII, FcγRIII) and/or the complement proteinC1q.

In one embodiment, antibodies of the invention comprise an Fc regioncomprising at least one amino acid substitution at position Asn297,wherein the numbering system of the heavy chain is that of the EU indexas in Kabat. In a specific embodiment, antibodies of the inventioncomprise the amino acid substitution Asn297Asp in the heavy chainregion, wherein induction of effector function is reduced compared toeffector function induced by an antibody comprising the parentnon-substituted antibody heavy chain region. In a further embodiment,antibodies of the invention comprise an Fc region comprising at leastone amino acid substitution at position Pro329, wherein the numberingsystem of the heavy chain is that of the EU index as in Kabat. In aspecific embodiment, antibodies of the invention comprise the amino acidsubstitution Pro329Gly in the heavy chain region, wherein induction ofeffector function is reduced compared to effector function induced by anantibody comprising the parent non-substituted antibody heavy chainregion. In a further embodiment said antibodies comprise at least one ormore of the following properties: reduced or abolished effector (ADCCand/or CDC and/or ADCP) function, reduced or abolished binding to Fcreceptors, reduced or abolished binding to C1q and reduced or abolishedinfusion reaction (cytokine release syndrome).

Accordingly, in one embodiment, an antibody is provided, comprising atleast one amino acid substitution relative to the parent non-substitutedheavy chain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residue Asn297, andwherein said substitution is Asn297Asp, wherein induction of effectorfunction is reduced compared to effector function induced by an antibodycomprising the parent non-substituted heavy chain region, whereinFcγRIII binding by the antibody comprising the variant heavy chainregion is abolished compared to binding to FcγRIII by the parentnon-substituted antibody comprising asparagine at position 297. In afurther embodiment, an antibody as described herein is provided, whereinADCC function induced by the antibody comprising the variant heavy chainregion is abolished or strongly reduced compared to ADCC functioninduced by the parent non-substituted antibody comprising asparagine atposition 297. In a further embodiment, an antibody as described hereinis provided, wherein FcγRI binding by the antibody comprising thevariant heavy chain region is reduced compared to binding to FcγRI bythe parent non-substituted antibody comprising asparagine at position297. In yet a further embodiment, an antibody as described herein isprovided, wherein induction of ADCP function induced by the antibodycomprising the variant heavy chain region is reduced compared to ADCPfunction induced by the parent non-substituted antibody comprisingasparagine at position 297, wherein the antibody comprising the variantheavy chain retains residual ADCP function. In yet a further embodiment,an antibody as described herein is provided, wherein induction of CDCfunction induced by the antibody comprising the variant heavy chainregion is abolished compared to CDC function induced by the parentnon-substituted antibody comprising asparagine at position 297. In afurther embodiment, an antibody is provided, comprising at least oneamino acid substitution relative to the parent non-substituted heavychain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residue Pro329, andwherein said substitution is Pro329Gly, wherein induction of effectorfunction is reduced compared to effector function induced by an antibodycomprising the parent non-substituted heavy chain region, whereinFcγRIII binding by the antibody comprising the variant heavy chainregion is abolished compared to binding to FcγRIII by the parentnon-substituted antibody comprising proline at position 329. In afurther embodiment, an antibody as described herein is provided, whereinADCC function induced by the antibody comprising the variant heavy chainregion is abolished or strongly reduced compared to ADCC functioninduced by the parent non-substituted antibody comprising proline atposition 329. In a further embodiment, an antibody as described hereinis provided, wherein FcγRI binding by the antibody comprising thevariant heavy chain region is reduced compared to binding to FcγRI bythe parent non-substituted antibody comprising proline at position 329.In yet a further embodiment, an antibody as described herein isprovided, wherein induction of ADCP function induced by the antibodycomprising the variant heavy chain region is reduced compared to ADCPfunction induced by the parent non-substituted antibody comprisingproline at position 329, wherein the antibody comprising the variantheavy chain retains residual ADCP function. In yet a further embodiment,an antibody as described herein is provided, wherein induction of CDCfunction induced by the antibody comprising the variant heavy chainregion is abolished compared to CDC function induced by the parentnon-substituted antibody comprising prolilne at position 329.

In still another embodiment, the heavy chain variants of the presentinvention exhibit a reduced affinity to a human Fc receptor (FcγR)and/or a human complement receptor as compared to the parent antibodycomprising the wildtype Fc region. In another embodiment, said antibodycomprising a variant heavy chain region exhibits a reduced affinity to ahuman Fc receptor (FcγR) and/or a human complement receptor as comparedto the parent antibody comprising the wildtype human Fc region. In afurther embodiment the affinity to at least one of the FcγRI, FcγRII,FcγRIII is reduced, in a still further embodiment the affinity to theFcγRI and FcγRIII is reduced, and in a still further embodiment theaffinity to the FcγRI, FcγRII and FcγRIII is reduced, in still a furtheraspect of the invention the affinity to the FcγRI receptor, FcγRIIIreceptor and C1q is reduced, and in still a further aspect of theinvention the affinity to the FcγRI, FcγRII, FcγRIII and C1q receptor isreduced. In still a further embodiment the ADCC induced by said antibodycomprising a heavy chain variant is reduced. In still a further aspectof the invention, the ADCC and CDC induced by the antibody comprisingthe wildtype Fc polypeptide is reduced or abolished. In a still furtheraspect the antibody comprising an Fc variant disclosed herein exhibit adecreased ADCC, CDC and ADCP compared to the parent antibody comprisingthe wildtype Fc polypeptide. In yet a further embodiment the presentinvention is directed to antibodies comprising the amino acidsubstitution Asn297Asp in the Fc region of the antibodies leading toreduced or abolished infusion reaction (cytokine release syndrome)compared to the parent non-substituted antibody. In yet a furtherembodiment the present invention is directed to antibodies comprisingthe amino acid substitution Pro329Gly in the Fc region of the antibodiesleading to reduced or abolished infusion reaction (cytokine releasesyndrome) compared to the parent non-substituted antibody. In anotherspecific embodiment, the parent non-substituted antibody isobinutuzumab. In another specific embodiment, the parent non-substitutedantibody is rituximab.

In a further aspect, the present invention is directed to antibodieswith modified Fc region resulting with reduced effector function asdescribed herein further comprising a modified heavy chain CH1 and/or VHregion, whereby the ability of these antibodies to induce cell signalingactivity of a target antigen and/or mediate cross-linking of targetantigen can be enhanced (i.e., induced or increased) or reduced (i.e.,inhibited or decreased). In a further aspect, said antibodies comprise amodified Fc region, whereby the induction of antibody dependentcell-mediated cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP) and complement dependent cytotoxicity (CDC) isreduced and a modified CH1 region wherein induction of direct cell deathis altered. In a further aspect, said antibodies comprise a modified Fcregion, whereby the induction of antibody dependent cell-mediatedcytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) andcomplement dependent cytotoxicity (CDC) is reduced and a modified VHregion wherein induction of direct cell death is altered. In a furtheraspect said modification to the Fc region leads to reduced but notabolished induction of ADCP function. In yet a further aspect anantibody modified according to the invention retains residual ADCPfunction.

In one embodiment the present invention is directed to antibodies asdescribed herein having a modification at position Asn297 in the Fcregion of the antibodies resulting with reduced induction of antibodydependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP) and complement dependent cytotoxicity (CDC), saidantibodies further comprising a modification at position Pro151 in theCH1 region and/or a modification at Leu11 in the VH region, resultingwith altered signaling behavior of the antibodies. In one embodiment thepresent invention is directed to antibodies as described herein having amodification at position Pro329 in the Fc region of the antibodiesresulting with reduced induction of antibody dependent cell-mediatedcytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) andcomplement dependent cytotoxicity (CDC), said antibodies furthercomprising a modification at position Pro151 in the CH1 region and/or amodification at Leu11 in the VH region, resulting with altered signalingbehavior of the antibodies. In certain embodiments, antibodiescomprising a variant Fc and/or CH1 and/or VH of the invention compriseat least one or more of the following properties: increased or decreasedinduction of direct cell death, reduced or abolished effector (ADCCand/or CDC and/or ADCP) function, reduced or abolished binding to Fcreceptors, reduced or abolished binding to C1q and reduced or abolishedinfusion reaction (cytokine release syndrome). More specifically,embodiments of the invention provide anti-CD20 antibodies with increasedinduction of direct cell death and reduced affinity for Fc receptors(e.g., FcγRI, FcγRII, FcγRIII).

The modified heavy chain CH1, VH and Fc regions of the antibodies of thepresent invention differ from the corresponding non-substituted parentpolypeptide regions by at least one amino acid substitution. The“parent”, “starting”, “nonmodified” or “non-substituted” polypeptidepreferably comprises at least a portion of an antibody heavy chainregion, and can be prepared using techniques available in the art forgenerating polypeptides comprising an Fc region as well as a heavy chainCH1 and VH region or portions thereof.

Accordingly, in one embodiment, an antibody is provided, comprising avariant heavy chain region comprising at least one amino acidsubstitution relative to the parent non-substituted heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residue Asn297, and wherein said substitutionis Asn297Asp, wherein induction of effector function is reduced comparedto effector function induced by an antibody comprising the parentnon-substituted heavy chain region, wherein the variant heavy chainregion comprises a further amino acid substitution relative to theparent non-substituted heavy chain region, wherein the heavy chainregion of the parent non-substituted antibody comprises the amino acidresidue Pro151, and wherein said further substitution is at said aminoacid residue Pro151, wherein direct cell death induced by the antibodycomprising the variant heavy chain region is altered compared to directcell death induced by the antibody comprising proline at position 151.Accordingly, in one embodiment, an antibody is provided, comprising avariant heavy chain region comprising at least one amino acidsubstitution relative to the parent non-substituted heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residue Pro329, and wherein said substitutionis Pro329Gly, wherein induction of effector function is reduced comparedto effector function induced by an antibody comprising the parentnon-substituted heavy chain region, wherein the variant heavy chainregion comprises a further amino acid substitution relative to theparent non-substituted heavy chain region, wherein the heavy chainregion of the parent non-substituted antibody comprises the amino acidresidue Pro151, and wherein said further substitution is at said aminoacid residue Pro151, wherein direct cell death induced by the antibodycomprising the variant heavy chain region is altered compared to directcell death induced by the antibody comprising proline at position 151.In another embodiment, the antibody as described herein is provided,wherein direct cell death induced by the antibody comprising the variantheavy chain region is increased compared to direct cell death induced bythe antibody comprising proline at position 151. In yet anotherembodiment, the antibody as described herein is provided, wherein directcell death induced by the antibody comprising the variant heavy chainregion is decreased compared to direct cell death induced by theantibody comprising proline at position 151. In yet another embodiment,the antibody as described herein is provided, wherein Pro151 issubstituted with an amino acid selected from the group consisting ofalanine and phenylalanine. In yet another embodiment, the antibody asdescribed herein is provided, wherein Pro151 is substituted withphenylalanine, wherein direct cell death induced by the antibodycomprising the variant heavy chain region is increased compared todirect cell death induced by the antibody comprising proline at position151. In yet another embodiment, the antibody as described herein isprovided, wherein Pro151 is substituted with alanine, wherein directcell death induced by the antibody comprising the variant heavy chainregion is decreased compared to direct cell death induced by theantibody comprising proline at position 151. In another specificembodiment, the parent non-substituted antibody is obinutuzumab and saidvariant heavy chain region comprises the amino acid substitutionsPro151Phe of the CH1 region and Asn297Asp of the Fc region relative toobinutuzumab. In another specific embodiment, the parent non-substitutedantibody is obinutuzumab and said variant heavy chain region comprisesthe amino acid substitutions Pro151Phe of the CH1 region and Pro329Glyof the Fc region relative to obinutuzumab. In another specificembodiment, the parent non-substituted antibody is rituximab and saidvariant heavy chain region comprises the amino acid substitutionsPro151Phe of the CH1 region and Asn297Asp of the Fc region relative torituximab. In another specific embodiment, the parent non-substitutedantibody is rituximab and said variant heavy chain region comprises theamino acid substitutions Pro151Phe of the CH1 region and Pro329Gly ofthe Fc region relative to rituximab.

In a further embodiment, an antibody is provided, comprising a variantheavy chain region comprising at least one amino acid substitutionrelative to the parent non-substituted heavy chain region, wherein theheavy chain region of the parent non-substituted antibody comprises theamino acid residue Asn297, and wherein said substitution is Asn297Asp,wherein induction of effector function is reduced compared to effectorfunction induced by an antibody comprising the parent non-substitutedheavy chain region, wherein the variant heavy chain region comprises afurther amino acid substitution relative to the parent non-substitutedheavy chain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residue Val11, andwherein said further substitution is at said amino acid residue Val11,wherein direct cell death induced by the antibody comprising the variantheavy chain region is altered compared to direct cell death induced bythe antibody comprising valine at position 11. In a further embodiment,an antibody is provided, comprising a variant heavy chain regioncomprising at least one amino acid substitution relative to the parentnon-substituted heavy chain region, wherein the heavy chain region ofthe parent non-substituted antibody comprises the amino acid residuePro329, and wherein said substitution is Pro329Gly, wherein induction ofeffector function is reduced compared to effector function induced by anantibody comprising the parent non-substituted heavy chain region,wherein the variant heavy chain region comprises a further amino acidsubstitution relative to the parent non-substituted heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residue Val11, and wherein said furthersubstitution is at said amino acid residue Val11, wherein direct celldeath induced by the antibody comprising the variant heavy chain regionis altered compared to direct cell death induced by the antibodycomprising valine at position 11. In another embodiment, the antibody asdescribed herein is provided, wherein direct cell death induced by theantibody comprising the variant heavy chain region is increased comparedto direct cell death induced by the antibody comprising valine atposition 11. In yet another embodiment, the antibody as described hereinis provided, wherein direct cell death induced by the antibodycomprising the variant heavy chain region is decreased compared todirect cell death induced by the antibody comprising valine at position11. In yet another embodiment, the antibody as described herein isprovided, wherein Val11 is substituted with an amino acid selected fromthe group consisting of alanine, glycine, phenylalanine, threonine andtryptophan. In yet another embodiment, the antibody as described hereinis provided, wherein Val11 is substituted with an amino acid selectedfrom the group consisting of phenylalanine, threonine and tryptophan,wherein direct cell death induced by the antibody comprising the variantheavy chain region is increased compared to direct cell death induced bythe antibody comprising valine at position 11. In yet anotherembodiment, the antibody as described herein is provided, wherein Val11is substituted with an amino acid selected from the group consisting ofalanine and glycine, wherein direct cell death induced by the antibodycomprising the variant heavy chain region is decreased compared todirect cell death induced by the antibody comprising valine at positionVal11. In another specific embodiment, the parent non-substitutedantibody is obinutuzumab and said variant heavy chain region comprisesthe amino acid substitutions Val11Phe of the VH region and Asn297Asp ofthe Fc region relative to obinutuzumab. In another specific embodiment,the parent non-substituted antibody is rituximab and said variant heavychain region comprises the amino acid substitutions Val11Phe of the VHregion and Asn297Asp of the Fc region relative to rituximab. In anotherspecific embodiment, the parent non-substituted antibody is obinutuzumaband said variant heavy chain region comprises the amino acidsubstitutions Val11Phe of the VH region and Pro329Gly of the Fc regionrelative to obinutuzumab. In another specific embodiment, the parentnon-substituted antibody is rituximab and said variant heavy chainregion comprises the amino acid substitutions Val11Phe of the VH regionand Pro329Gly of the Fc region relative to rituximab.

Accordingly, the amino acid sequence of the parent polypeptide can bemodified according to the invention to generate an antibody having amodification at position Asn297 and/or Pro329 in the Fc region, withreduced ability to induce effector function, as well as a modificationat position Pro151 in the heavy chain CH1 and/or position Leu11 in theheavy chain VH region, which results with altered ability to induce cellsignaling activity of a target antigen when the modified antibody iscomplexed with (e.g., bound to) the target antigen. The cell signalingactivity can be agonist activity or antagonist activity. According toone aspect of the invention, agonist activity is induced by a modifiedantigen binding molecule when it binds to a cell membrane-associatedreceptor and initiates a cell signaling pathway. In a specificembodiment, the cell signaling pathway is an apoptosis pathway. Inanother embodiment, the cell signaling pathway is a cell differentiationpathway. According to another aspect of the invention, antagonistactivity by a modified antigen binding molecule occurs, for example,when the antibody binds to a cell membrane-associated receptor andprevents the induction of a cell signaling pathway or disrupts anongoing signal. Antagonist activity can be achieved, for example, byblocking the binding and subsequent signal transduction of an endogenousligand and/or by preventing the cross-linking or oligomerization ofreceptors or other molecules that would be necessary for induction of acell signaling pathway. In one embodiment, the cell signaling pathwaythat is inhibited or disrupted is a cell growth pathway. In anotherembodiment, the cell signaling pathway that is inhibited or disrupted isa cell division pathway. In another embodiment the cell signalingpathway that is inhibited or disrupted is a cell survival pathway.Likewise, the amino acid sequence of the parent polypeptide can also bemodified to generate an antibody according to the present inventionhaving a modification at position Asn297 and/or Pro329 in the Fc region,with reduced ability to induce effector function, as well as amodification at position Pro151 in the heavy chain CH1 and/or positionLeu11 in the heavy chain VH region, with altered ability to mediatecross-linking of one or more target antigens when the modified antibodyis complexed with (e.g., bound to) the target antigen(s). In oneembodiment, the bound target antigens (e.g., cell surface receptormolecules) are brought into closer proximity to each other and/or a morefavorable conformation for interaction than they would be by thecorresponding non-substituted parent antibody, thereby increasingcross-linking and oligomerization between the bound antigens. In anotherembodiment, the bound target antigens (e.g., cell surface receptormolecules) are kept farther apart from each other, and/or in a lessfavorable conformation for interaction than they would be by thecorresponding non-substituted parent antibody, thereby reducing orpreventing cross-linking and oligomerization between the bound antigens.In a particular embodiment, the increased cross-linking oroligomerization results in increased direct cell death. In anotherembodiment, the increased cross-linking or oligomerization results inincreased cell differentiation. In another embodiment, the reduction incross-linking or oligomerization results in decreased cell growth,decreased cell division, or decreased cell survival.

In certain specific embodiments the present invention is directed tomodified antibodies that have decreased ability to induce effectorfunction and increased ability to induce apoptosis compared to thecorresponding non-modified parent antibody. For example, a parentantibody that has little or no ability to induce apoptosis but strongability to induce effector function can be modified according to thepresent invention to generate a modified antibody that does have theability to induce apoptosis or that has an increased ability to induceapoptosis and that does no have the ability to induce strong effectorfunction. Likewise a parent antibody that induces strong effectorfunction can be modified to generate a modified antibody that inducesweak effector function while retaining or increasing the potential toinduce apoptosis. The present invention is also directed to modifiedantibodies that have increased ability to induce growth arrest or celldifferentiation and reduced or abolished induction of effector functionor cytokine release activity as compared to the correspondingnon-modified parent antibody. For example, a parent antibody that haslittle or no ability to induce growth arrest or cell differentiation canbe modified according to the present invention to generate a modifiedantibody that does have the ability to induce growth arrest ordifferentiation or that has an increased ability to induce growth arrestor differentiation while reducing adverse events like e.g., infusionreaction (cytokine release syndrome).

A further aspect of the present invention is the provision of modifiedanti-CD20 antibodies. In a preferred embodiment, the antibody asdisclosed herein specifically binds to CD20. In certain embodiments, themodified anti-CD20 antibody as disclosed herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM, from 10⁻⁸M to 10⁻¹³M or from 10⁻⁹M to 10⁻¹³ M. In a preferredembodiment, the antibody as disclosed herein binds to CD20 with adissociation constant (Kd) on cells of 10 nM or less as determined byscatchard analysis. In one embodiment, the present invention is directedto a modified anti-CD20 antibody comprising at least two amino acidsubstitutions in the heavy chain region compared to a type I parentanti-CD20 antibody, wherein the substitutions result in decreasedinduction of effector function and increased induction of direct celldeath by the modified anti-CD20 antibody. In another embodiment, thepresent invention is directed to modified type II anti-CD20 antibodieshaving decreased induction of effector function without loss ofsubstantial ability to induce direct cell death as a result of aminoacid substitutions as disclosed herein. In one embodiment, the type IIanti-CD20 antibodies comprise a substitution in two or more amino acidsin the heavy chain compared to a parent molecule. In another embodiment,the present invention is directed to a modified anti-CD20 antibody,comprising a variant heavy chain region, wherein the heavy chain regionof the parent non-substituted antibody comprises the amino acid residuesPro151 and Asn297, said variant heavy chain region comprising the aminoacid substitutions Asn297Asp and Pro151Phe relative to the parentnon-substituted heavy chain region, wherein effector function and directcell death induced by the antibody comprising the variant heavy chainregion is altered compared to effector function and direct cell deathinduced by the antibody comprising the parent non-substituted heavychain region. In another embodiment, the present invention is directedto a modified anti-CD20 antibody, comprising a variant heavy chainregion, wherein the heavy chain region of the parent non-substitutedantibody comprises the amino acid residues Pro151 and Pro329, saidvariant heavy chain region comprising the amino acid substitutionsPro329Gly and Pro151Phe relative to the parent non-substituted heavychain region, wherein effector function and direct cell death induced bythe antibody comprising the variant heavy chain region is alteredcompared to effector function and direct cell death induced by theantibody comprising the parent non-substituted heavy chain region. Inanother embodiment, direct cell death induced by the antibody comprisingthe variant heavy chain region according to the invention is increasedcompared to direct cell death induced by the antibody comprising theparent non-substituted heavy chain region. In another embodiment, directcell death induced by the antibody comprising the variant heavy chainregion according to the invention is decreased compared to direct celldeath induced by the antibody comprising the parent non-substitutedheavy chain region. In another embodiment, effector functions induced bythe antibody comprising the variant heavy chain region according to theinvention are reduced or abolished compared to effector functionsinduced by the antibody comprising the parent non-substituted heavychain region. In a preferred embodiment, direct cell death induced bythe antibody comprising the variant heavy chain region according to theinvention is increased compared to direct cell death induced by theantibody comprising the parent non-substituted heavy chain region andeffector function induced by the antibody comprising the variant heavychain region according to the invention is reduced or abolished comparedto direct cell death induced by the antibody comprising the parentnon-substituted heavy chain region. In a further preferred embodimentthe antibody according to the invention, comprising said amino acidsubstitution Asn297Asp or Pro329Gly, retains residual ADCP function.

In a further embodiment of the present invention, a modified anti-CD20antibody is provided, comprising a variant CH1 and/or VH region asdisclosed herein and a variant Fc region as disclosed herein compared tothe respective parent non-substituted antibody. In one embodiment ananti-CD20 antibody is provided, comprising a variant heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residues Pro151 and Asn297, said variant heavychain region comprising the amino acid substitutions Pro151Phe andAsn297Asp relative to the parent non-substituted heavy chain region,wherein induction of direct cell death is increased, and wherein FcγRIIIbinding is abolished, wherein induction of ADCC is abolished or stronglyreduced, wherein FcγRI binding is reduced, and wherein induction of ADCPfunction is reduced but residual ADCP function is preserved, compared todirect cell death and effector function induced by an antibodycomprising the parent non-substituted antibody heavy chain region. Inone embodiment an anti-CD20 antibody is provided, comprising a variantheavy chain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residues Pro151 andPro329, said variant heavy chain region comprising the amino acidsubstitutions Pro151Phe and Pro329Gly relative to the parentnon-substituted heavy chain region, wherein induction of direct celldeath is increased, and wherein FcγRIII binding is abolished, whereininduction of ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, and wherein induction of ADCP function is reducedbut residual ADCP function is preserved, compared to direct cell deathand effector function induced by an antibody comprising the parentnon-substituted antibody heavy chain region. In a further embodiment ananti-CD20 antibody is provided, comprising a variant heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residues Val11 and Asn297, said variant heavychain region comprising the amino acid substitutions Val11Phe andAsn297Asp relative to the parent non-substituted heavy chain region,wherein induction of direct cell death is increased, and wherein FcγRIIIbinding is abolished, wherein induction of ADCC is abolished or stronglyreduced, wherein FcγRI binding is reduced, and wherein induction of ADCPfunction is reduced but residual ADCP function is preserved, compared todirect cell death and effector function induced by an antibodycomprising the parent non-substituted antibody heavy chain region. In afurther embodiment an anti-CD20 antibody is provided, comprising avariant heavy chain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residues Val11 andPro329, said variant heavy chain region comprising the amino acidsubstitutions Val11 Phe and Pro329Gly relative to the parentnon-substituted heavy chain region, wherein induction of direct celldeath is increased, and wherein FcγRIII binding is abolished, whereininduction of ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, and wherein induction of ADCP function is reducedbut residual ADCP function is preserved, compared to direct cell deathand effector function induced by an antibody comprising the parentnon-substituted antibody heavy chain region. In a further aspect saidantibodies exhibit a reduced affinity to the human FcγRIII and/or FcγRIIand/or FcγRI compared to antibodies with wildtype Fc region. In afurther aspect, said modifications in the Fc region lead to reduced orabolished effector (ADCC and/or CDC and/or ADCP) function, reduced orabolished binding to Fc receptors, reduced or abolished binding to C1qand reduced or abolished infusion reaction (cytokine release syndrome).

In a more specific embodiment, the present invention provides anti-CD20antibodies with increased induction of direct cell death and reducedaffinity to a human Fc receptor (FcγR) and/or a human complementreceptor as compared to the polypeptide comprising the wildtype humanheavy chain region. In a further embodiment the affinity to at least oneof the FcγRI, FcγRII, FcγRIII is reduced, in a still further embodimentthe affinity to the FcγRI and FcγRIII is reduced, and in a still furtherembodiment the affinity to the FcγRI, FcγRII and FcγRIII is reduced, instill a further aspect of the invention the affinity to the FcγRIreceptor, FcγRIII receptor and C1q is reduced, and in still a furtheraspect of the invention the affinity to the FcγRI, FcγRII, FcγRIII andC1q receptor is reduced.

In another aspect of the present invention, antibodies are provided,comprising at least one amino acid substitution at one of the amino acidresidues selected from the group consisting of Val11, Pro151 and Asn297,wherein induction of effector function and/or binding to Fcγ receptorsand induction of direct cell death by the antibody comprising thevariant heavy chain region is altered. In another aspect of the presentinvention, antibodies are provided, comprising at least one amino acidsubstitution at one of the amino acid residues selected from the groupconsisting of Val11, Pro151, Asn297 and Pro329, wherein induction ofeffector function and/or binding to Fcγ receptors and induction ofdirect cell death by the antibody comprising the variant heavy chainregion is altered. In one embodiment, binding to FcγRIII by the antibodycomprising the variant heavy chain region is reduced. In preferredembodiments, binding to FcγRIII by the antibody comprising the variantheavy chain region is reduced to 90% or less, 80% or less, 70% or less,to 60% or less, to 50% or less, to 40% or less, to 30% or less, to 20%or less, to 10% or less of FcγRIII binding by the antibody comprisingthe parent non-substituted heavy chain region. In a more preferredembodiment, binding to FcγRIII by the antibody comprising the variantheavy chain region is reduced to 0% to 20% of FcγRIII binding by theantibody comprising the parent non-substituted heavy chain region. In amost preferred embodiment, binding to FcγRIII by the antibody comprisingthe variant heavy chain region is abolished compared to binding toFcγRIII by the antibody comprising the parent non-substituted heavychain region. In another embodiment, induction of ADCC function by theantibody comprising the variant heavy chain region is reduced. Inpreferred embodiments, induction of ADCC function by the antibodycomprising the variant heavy chain region is reduced to 90% or less, 80%or less, 70% or less, to 60% or less, to 50% or less, to 40% or less, to30% or less, to 20% or less, to 10% or less of ADCC function induced bythe antibody comprising the parent non-substituted heavy chain region.In a more preferred embodiment, induction of ADCC function by theantibody comprising the variant heavy chain region is reduced to 0% to80%, and in yet a more preferred embodiment to 0% to 20% of ADCCfunction induced by the antibody comprising the parent non-substitutedheavy chain region. In a most preferred embodiment, induction of ADCCfunction by the antibody comprising the variant heavy chain region isabolished compared to ADCC function induced by the antibody comprisingthe parent non-substituted heavy chain region. In a further embodiment,ADCC may be measured by quantification of LDH released into cellsupernatants in the presence of effector cells. In yet anotherembodiment, binding to FcγRI by the antibody comprising the variantheavy chain region is reduced. In another embodiment, binding to FcγRIby the antibody comprising the variant heavy chain region is abolishedcompared to binding to FcγRI by the antibody comprising the parentnon-substituted heavy chain region. In preferred embodiments, binding toFcγRI by the antibody comprising the variant heavy chain region isreduced to 90% or less, 80% or less, 70% or less, to 60% or less, to 50%or less, to 40% or less, to 30% or less, to 20% or less, to 10% or lessof FcγRI binding by the antibody comprising the parent non-substitutedheavy chain region. In a more preferred embodiment, binding to FcγRI bythe antibody comprising the variant heavy chain region is reduced to 10%to 90%, and in a most preferred embodiment to 20% to 60% of FcγRIbinding by the antibody comprising the parent non-substituted heavychain region. In another embodiment, induction of ADCP function by theantibody comprising the variant heavy chain region is reduced. In afurther embodiment, induction of ADCP function by the antibodycomprising the variant heavy chain region is abolished compared to ADCPfunction induced by the antibody comprising the parent non-substitutedheavy chain region. In preferred embodiments, induction of ADCP functionby the antibody comprising the variant heavy chain region is reduced to90% or less, 80% or less, 70% or less, to 60% or less, to 50% or less,to 40% or less, to 30% or less, to 20% or less, to 10% or less of ADCPfunction induced by the antibody comprising the parent non-substitutedheavy chain region. In a more preferred embodiment, induction of ADCPfunction by the antibody comprising the variant heavy chain region isreduced to 10% to 90%, and in yet a more preferred embodiment to 20% to60% of ADCP function induced by the antibody comprising the parentnon-substituted heavy chain region. In a most preferred embodiment,antibodies of the present invention preserve residual ADCP function. Ina further embodiment, ADCP may be measured by quantification ofCFSE-PKH26-double positive macrophages after co-incubation with targetcells. In another embodiment, induction of CDC function by the antibodycomprising the variant heavy chain region is reduced. In preferredembodiments, induction of CDC function by the antibody comprising thevariant heavy chain region is reduced to 90% or less, 80% or less, 70%or less, to 60% or less, to 50% or less, to 40% or less, to 30% or less,to 20% or less, to 10% or less of CDC function induced by the antibodycomprising the parent non-substituted heavy chain region. In a morepreferred embodiment, induction of CDC function by the antibodycomprising the variant heavy chain region is reduced to 0% to 80%, andin yet a more preferred embodiment to 0% to 20% of CDC function inducedby the antibody comprising the parent non-substituted heavy chainregion. In a most preferred embodiment, induction of CDC function by theantibody comprising the variant heavy chain region is abolished comparedto CDC function induced by the antibody comprising the parentnon-substituted heavy chain region. In a further embodiment, CDC may bemeasured by quantification of LDH released into cell supernatants in thepresence of complement. In another embodiment induction of infusionreaction (cytokine release syndrome) by the antibody comprising thevariant heavy chain region is reduced. In preferred embodiments,induction of infusion reaction by the antibody comprising the variantheavy chain region is reduced to 90% or less, 80% or less, 70% or less,to 60% or less, to 50% or less, to 40% or less, to 30% or less, to 20%or less, to 10% or less of infusion reaction induced by the antibodycomprising the parent non-substituted heavy chain region. In a mostpreferred embodiment, induction of infusion reaction by the antibodycomprising the variant heavy chain region is abolished.

Accordingly, in a further embodiment, direct cell death induced by theantibody comprising the variant heavy chain region is increased comparedto direct cell death induced by the antibody comprising the parentnon-substituted heavy chain region. In another aspect of the presentinvention, direct cell death induced by the antibody comprising thevariant heavy chain region is increased to at least 110% of the directcell death induced by the antibody comprising the parent non-substitutedheavy chain region. In a preferred embodiment, direct cell death inducedby the antibody comprising the variant heavy chain region is increasedto at least 120% of the direct cell death induced by the antibodycomprising the parent non-substituted heavy chain region. In morepreferred embodiments, direct cell death induced by the antibodycomprising the variant heavy chain region is increased to at least 130%,to at least 140%, to at least 150%, to at least 160%, to at least 170%,to at least 180%, to at least 190% to at least 200% of the direct celldeath induced by the antibody comprising the parent non-substitutedheavy chain region. In a most preferred embodiment, direct cell deathinduced by the antibody comprising the variant heavy chain region isincreased to 120% to 200% of the direct cell death induced by theantibody comprising the parent non-substituted heavy chain region. Inyet a further aspect of the present invention, direct cell death inducedby the antibody comprising the variant heavy chain region is decreasedcompared to direct cell death induced by the antibody comprising theparent non-substituted heavy chain region. In still a furtherembodiment, direct cell death induced by the antibody comprising thevariant heavy chain region is decreased to 90% or less of the directcell death induced by the antibody comprising the parent non-substitutedheavy chain region. In a preferred embodiment, direct cell death inducedby the antibody comprising the variant heavy chain region is decreasedto 80% or less of the direct cell death induced by the antibodycomprising the parent non-substituted heavy chain region. In morepreferred embodiments, direct cell death induced by the antibodycomprising the variant heavy chain region is decreased to 70% or less,to 60% or less, to 50% or less, to 40% or less, to 30% or less, to 20%or less, to 10% or less of the direct cell death induced by the antibodycomprising the parent non-substituted heavy chain region. In anothermore preferred embodiment, direct cell death induced by the antibodycomprising the variant heavy chain region is decreased to 0% to 70%, andin yet a more preferred embodiment to 0% to 20% of the direct cell deathinduced by the antibody comprising the parent non-substituted heavychain region. In still another embodiment, direct cell death induced bythe antibody comprising the variant heavy chain region is abolished. Ina further embodiment induction of direct cell death may be measured byAnnexin V binding and PI staining.

While, it is preferred to alter binding to a FcγR, Fc region variantswith altered binding affinity for the neonatal receptor (FcRn) are alsocontemplated herein. Fc region variants with improved affinity for FcRnare anticipated to have longer serum half-lives, and such molecules willhave useful applications in methods of treating mammals where longhalf-life of the administered polypeptide is desired, e.g., to treat achronic disease or disorder. Fc region variants with decreased FcRnbinding affinity, on the contrary, are expected to have shorterhalf-lives, and such molecules may, for example, be administered to amammal where a shortened circulation time may be advantageous, e.g. forin vivo diagnostic imaging or for polypeptides which have toxic sideeffects when left circulating in the blood stream for extended periods,etc. Fc region variants with decreased FcRn binding affinity areanticipated to be less likely to cross the placenta, and thus may beutilized in the treatment of diseases or disorders in pregnant women. Fcregion variants with altered binding affinity for FcRn include thosecomprising an Fc region amino acid modification at any one or more ofamino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288,303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380,382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 or 447. Thosewhich display reduced binding to FcRn will generally comprise an Fcregion amino acid modification at any one or more of amino acidpositions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435,436, 439 or 447; and those with increased binding to FcRn will usuallycomprise an Fc region amino acid modification at any one or more ofamino acid positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434.

In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residue Asn297,said variant heavy chain region comprising the amino acid substitutionAsn297Asp relative to the parent non-substituted heavy chain region,wherein FcγRIII binding is abolished, wherein ADCC is abolished orstrongly reduced, wherein FcγRI binding is reduced, wherein induction ofADCP function is reduced, compared to effector function induced by theparent non-substituted antibody comprising asparagine at position 297.In a further specific embodiment, an anti-CD20 antibody is provided,comprising a variant heavy chain region, wherein the heavy chain regionof the parent non-substituted antibody comprises the amino acid residuePro329, said variant heavy chain region comprising the amino acidsubstitution Pro329Gly relative to the parent non-substituted heavychain region, wherein FcγRIII binding is abolished, wherein ADCC isabolished or strongly reduced, wherein FcγRI binding is reduced, whereininduction of ADCP function is reduced, compared to effector functioninduced by the parent non-substituted antibody comprising proline atposition 329.

In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residue Asn297,said variant heavy chain region comprising the amino acid substitutionAsn297Asp relative to the parent non-substituted heavy chain region,wherein FcγRIII binding is abolished, wherein ADCC is abolished orstrongly reduced, wherein FcγRI binding is reduced, and whereininduction of ADCP function is reduced but residual ADCP function ispreserved, compared to effector function induced by the parentnon-substituted antibody comprising asparagine at position 297. In afurther specific embodiment, an anti-CD20 antibody is provided,comprising a variant heavy chain region, wherein the heavy chain regionof the parent non-substituted antibody comprises the amino acid residuePro329, said variant heavy chain region comprising the amino acidsubstitution Pro329Gly relative to the parent non-substituted heavychain region, wherein FcγRIII binding is abolished, wherein ADCC isabolished or strongly reduced, wherein FcγRI binding is reduced, andwherein induction of ADCP function is reduced but residual ADCP functionis preserved, compared to effector function induced by the parentnon-substituted antibody comprising proline at position 329.

In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residue Asn297,said variant heavy chain region comprising the amino acid substitutionAsn297Asp relative to the parent non-substituted heavy chain region,wherein FcγRIII binding is abolished, wherein ADCC is abolished orstrongly reduced, wherein FcγRI binding is reduced, wherein induction ofADCP function is reduced but residual ADCP function is preserved, andwherein infusion reaction (cytokine release syndrome) is reducedcompared to effector function induced by the parent non-substitutedantibody comprising asparagine at position 297. In a further specificembodiment, an anti-CD20 antibody is provided, comprising a variantheavy chain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residue Pro329, saidvariant heavy chain region comprising the amino acid substitutionPro329Gly relative to the parent non-substituted heavy chain region,wherein FcγRIII binding is abolished, wherein ADCC is abolished orstrongly reduced, wherein FcγRI binding is reduced, wherein induction ofADCP function is reduced but residual ADCP function is preserved, andwherein infusion reaction (cytokine release syndrome) is reducedcompared to effector function induced by the parent non-substitutedantibody comprising proline at position 329.

In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residues Pro151and Asn297, said variant heavy chain region comprising the amino acidsubstitutions Pro151Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by the parent non-substitutedantibody comprising asparagine at position 297, and wherein induction ofdirect cell death is increased compared to direct cell death induced bythe parent non-substituted antibody comprising proline at position 151.In another specific embodiment, an anti-CD20 antibody is provided,comprising a variant heavy chain region, wherein the heavy chain regionof the parent non-substituted antibody comprises the amino acid residuesPro151 and Pro329, said variant heavy chain region comprising the aminoacid substitutions Pro151Phe and Pro329Gly relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by the parent non-substitutedantibody comprising proline at position 329, and wherein induction ofdirect cell death is increased compared to direct cell death induced bythe parent non-substituted antibody comprising proline at position 151.

In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residues Pro151and Asn297, said variant heavy chain region comprising the amino acidsubstitutions Pro151Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, and wherein infusion reaction(cytokine release syndrome) is reduced compared to effector functioninduced by the parent non-substituted antibody comprising asparagine atposition 297, and wherein induction of direct cell death is increasedcompared to direct cell death induced by the parent non-substitutedantibody comprising proline at position 151. In another specificembodiment, an anti-CD20 antibody is provided, comprising a variantheavy chain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residues Pro151 andPro329, said variant heavy chain region comprising the amino acidsubstitutions Pro151Phe and Pro329Gly relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, and wherein infusion reaction(cytokine release syndrome) is reduced compared to effector functioninduced by the parent non-substituted antibody comprising proline atposition 329, and wherein induction of direct cell death is increasedcompared to direct cell death induced by the parent non-substitutedantibody comprising proline at position 151.

In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residues Pro151and Asn297, said variant heavy chain region comprising the amino acidsubstitutions Pro151Ala and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by the parent non-substitutedantibody comprising asparagine at position 297, and wherein induction ofdirect cell death is decreased compared to direct cell death induced bythe parent non-substituted antibody comprising proline at position 151.In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residues Pro151and Pro329, said variant heavy chain region comprising the amino acidsubstitutions Pro151Ala and Pro297Gly relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by the parent non-substitutedantibody comprising proline at position 329, and wherein induction ofdirect cell death is decreased compared to direct cell death induced bythe parent non-substituted antibody comprising proline at position 151.

In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residues Pro151and Asn297, said variant heavy chain region comprising the amino acidsubstitutions Pro151Ala and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced, andwherein infusion reaction (cytokine release syndrome) is reducedcompared to effector function induced by the parent non-substitutedantibody comprising asparagine at position 297, and wherein induction ofdirect cell death is decreased compared to direct cell death induced bythe parent non-substituted antibody comprising proline at position 151.

In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residues Val11and Asn297, said variant heavy chain region comprising the amino acidsubstitutions Val11Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by the parent non-substitutedantibody comprising asparagine at position 297, and wherein induction ofdirect cell death is increased compared to direct cell death induced bythe parent non-substituted antibody comprising valine at position 11. Ina specific embodiment, an anti-CD20 antibody is provided, comprising avariant heavy chain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residues Val11 andPro329, said variant heavy chain region comprising the amino acidsubstitutions Val11Phe and Pro329Gly relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by the parent non-substitutedantibody comprising proline at position 329, and wherein induction ofdirect cell death is increased compared to direct cell death induced bythe parent non-substituted antibody comprising valine at position 11.

In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residues Val11and Asn297, said variant heavy chain region comprising the amino acidsubstitutions Val11Ala and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by the parent non-substitutedantibody comprising asparagine at position 297, and wherein induction ofdirect cell death is decreased compared to direct cell death induced bythe parent non-substituted antibody comprising valine at position 11.

In a specific embodiment, an anti-CD20 antibody is provided, comprisinga variant heavy chain region, wherein the heavy chain region of theparent non-substituted antibody comprises the amino acid residues Val11and Asn297, said variant heavy chain region comprising the amino acidsubstitutions Val11Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, and wherein infusion reaction(cytokine release syndrome) is reduced compared to effector functioninduced by the parent non-substituted antibody comprising asparagine atposition 297, and wherein induction of direct cell death is increasedcompared to direct cell death induced by the parent non-substitutedantibody comprising valine at position 11.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residue Asn297 in the heavy chainregion, said variant heavy chain region comprising the amino acidsubstitution Asn297Asp relative to the parent non-substituted heavychain region, wherein FcγRIII binding is abolished, wherein ADCC isabolished or strongly reduced, wherein FcγRI binding is reduced, whereininduction of ADCP function is reduced, compared to effector functioninduced by obinutuzumab. In a specific embodiment, an antibody isprovided, comprising a variant heavy chain region, wherein the parentnon-substituted antibody is obinutuzumab comprising the amino acidresidue Pro329 in the heavy chain region, said variant heavy chainregion comprising the amino acid substitution Pro329GlyAsp relative tothe parent non-substituted heavy chain region, wherein FcγRIII bindingis abolished, wherein ADCC is abolished or strongly reduced, whereinFcγRI binding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residue Asn297 in the heavy chainregion, wherein the residues are numbered according to the EU index asin Kabat, said variant heavy chain region comprising the amino acidsubstitution Asn297Asp relative to the parent non-substituted heavychain region, wherein effector (ADCC and/or CDC and/or ADCP) functioninduced by the antibody comprising the variant heavy chain region isreduced or abolished compared to effector function induced byobinutuzumab, wherein infusion reaction (cytokine release syndrome)induced by the antibody comprising the variant heavy chain region isreduced or abolished compared to infusion reaction induced byobinutuzumab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residue Asn297 in the heavy chainregion, said variant heavy chain region comprising the amino acidsubstitution Asn297Asp relative to the parent non-substituted heavychain region, wherein FcγRIII binding is abolished, wherein ADCC isabolished or strongly reduced, wherein FcγRI binding is reduced, whereininduction of ADCP function is reduced but residual ADCP function ispreserved, compared to effector function induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a mutatedheavy chain, wherein the parent non-mutated antibody is obinutuzumabcomprising the amino acid residue Asn297 (EU numbering) in the heavychain, said mutated heavy chain comprising the amino acid substitutionAsn297Asp relative to the parent non-mutated heavy chain, whereinFcγRIII binding is abolished, wherein ADCC is abolished or stronglyreduced, wherein FcγRI binding is reduced, wherein induction of ADCPfunction is reduced but residual ADCP function is preserved, compared toeffector function induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a mutatedheavy chain, wherein the parent non-mutated antibody is obinutuzumabcomprising the amino acid residue Pro329 (EU numbering) in the heavychain, said mutated heavy chain comprising the amino acid substitutionPro329Gly relative to the parent non-mutated heavy chain, whereinFcγRIII binding is abolished, wherein ADCC is abolished or stronglyreduced, wherein FcγRI binding is reduced, wherein induction of ADCPfunction is reduced but residual ADCP function is preserved, compared toeffector function induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residues Pro151 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Pro151Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, compared to effector functioninduced by obinutuzumab, and wherein induction of direct cell death isincreased compared to direct cell death induced by obinutuzumab. In aspecific embodiment, an antibody is provided, comprising a variant heavychain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residues Pro151 and Pro329 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Pro151Phe and Pro329Gly relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, compared to effector functioninduced by obinutuzumab, and wherein induction of direct cell death isincreased compared to direct cell death induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residues Pro151 and Asn297 in theheavy chain region, wherein the residues are numbered according to theEU index as in Kabat, said variant heavy chain region comprising theamino acid substitutions Pro151Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein effector (ADCC and/or CDCand/or ADCP) function induced by the antibody comprising the variantheavy chain region is reduced or abolished compared to effector functioninduced by obinutuzumab, and wherein direct cell death induced by theantibody comprising the variant heavy chain region is increased comparedto direct cell death induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residues Pro151 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Pro151Ala and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by obinutuzumab, and whereininduction of direct cell death is decreased compared to direct celldeath induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residues Pro151 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Pro151Ala and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, compared to effector functioninduced by obinutuzumab, and wherein induction of direct cell death isdecreased compared to direct cell death induced by obinutuzumab. In aspecific embodiment, an antibody is provided, comprising a variant heavychain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residues Pro151 and Pro329 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Pro151Ala and Pro329Gly relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, compared to effector functioninduced by obinutuzumab, and wherein induction of direct cell death isdecreased compared to direct cell death induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residues Val11 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Val11Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by obinutuzumab, and whereininduction of direct cell death is increased compared to direct celldeath induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residues Val11 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Val11Ala and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by obinutuzumab, and whereininduction of direct cell death is decreased compared to direct celldeath induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residues Val11 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Val11Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, compared to effector functioninduced by obinutuzumab, and wherein induction of direct cell death isincreased compared to direct cell death induced by obinutuzumab. In aspecific embodiment, an antibody is provided, comprising a variant heavychain region, wherein the parent non-substituted antibody isobinutuzumab comprising the amino acid residues Val11 and Pro329 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Val11Phe and Pro329Gly relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, compared to effector functioninduced by obinutuzumab, and wherein induction of direct cell death isincreased compared to direct cell death induced by obinutuzumab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isrituximab comprising the amino acid residue Asn297 in the heavy chainregion, said variant heavy chain region comprising the amino acidsubstitution Asn297Asp relative to the parent non-substituted heavychain region, wherein FcγRIII binding is abolished, wherein ADCC isabolished or strongly reduced, wherein FcγRI binding is reduced, whereininduction of ADCP function is reduced, compared to effector functioninduced by rituximab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isrituximab comprising the amino acid residue Asn297 in the heavy chainregion, said variant heavy chain region comprising the amino acidsubstitution Asn297Asp relative to the parent non-substituted heavychain region, wherein effector (ADCC and/or CDC and/or ADCP) functioninduced by the antibody comprising the variant heavy chain region isreduced or abolished compared to effector function induced by rituximab,wherein infusion reaction (cytokine release syndrome) induced by theantibody comprising the variant heavy chain region is reduced orabolished compared to infusion reaction induced by rituximab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isrituximab comprising the amino acid residue Asn297 in the heavy chainregion, said variant heavy chain region comprising the amino acidsubstitution Asn297Asp relative to the parent non-substituted heavychain region, wherein FcγRIII binding is abolished, wherein ADCC isabolished or strongly reduced, wherein FcγRI binding is reduced, whereininduction of ADCP function is reduced but residual ADCP function ispreserved, compared to effector function induced by rituximab.

In a specific embodiment, an antibody is provided, comprising a mutatedheavy chain, wherein the parent non-mutated antibody is rituximabcomprising the amino acid residue Asn297 (EU numbering) in the heavychain, said mutated heavy chain comprising the amino acid substitutionAsn297Asp relative to the parent non-mutated heavy chain, whereinFcγRIII binding is abolished, wherein ADCC is abolished or stronglyreduced, wherein FcγRI binding is reduced, wherein induction of ADCPfunction is reduced but residual ADCP function is preserved, compared toeffector function induced by rituximab.

In a specific embodiment, an antibody is provided, comprising a mutatedheavy chain, wherein the parent non-mutated antibody is rituximabcomprising the amino acid residue Pro329 (EU numbering) in the heavychain, said mutated heavy chain comprising the amino acid substitutionPro329Gly relative to the parent non-mutated heavy chain, whereinFcγRIII binding is abolished, wherein ADCC is abolished or stronglyreduced, wherein FcγRI binding is reduced, wherein induction of ADCPfunction is reduced but residual ADCP function is preserved, compared toeffector function induced by rituximab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isrituximab comprising the amino acid residues Pro151 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Pro151Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, compared to effector functioninduced by rituximab, and wherein induction of direct cell death isincreased compared to direct cell death induced by rituximab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isrituximab comprising the amino acid residues Pro151 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Pro151Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein effector (ADCC and/or CDCand/or ADCP) function induced by the antibody comprising the variantheavy chain region is reduced or abolished compared to effector functioninduced by rituximab, and wherein direct cell death induced by theantibody comprising the variant heavy chain region is increased comparedto direct cell death induced by rituximab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isrituximab comprising the amino acid residues Pro151 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Pro151Ala and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by rituximab, and whereininduction of direct cell death is decreased compared to direct celldeath induced by rituximab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isrituximab comprising the amino acid residues Pro151 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Pro151Ala and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, compared to effector functioninduced by rituximab, and wherein induction of direct cell death isdecreased compared to direct cell death induced by rituximab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isrituximab comprising the amino acid residues Val11 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Val11 Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by rituximab, and whereininduction of direct cell death is increased compared to direct celldeath induced by rituximab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isrituximab comprising the amino acid residues Val11 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Val11Ala and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced,compared to effector function induced by rituximab, and whereininduction of direct cell death is decreased compared to direct celldeath induced by rituximab.

In a specific embodiment, an antibody is provided, comprising a variantheavy chain region, wherein the parent non-substituted antibody isrituximab comprising the amino acid residues Val11 and Asn297 in theheavy chain region, said variant heavy chain region comprising the aminoacid substitutions Val11Phe and Asn297Asp relative to the parentnon-substituted heavy chain region, wherein FcγRIII binding isabolished, wherein ADCC is abolished or strongly reduced, wherein FcγRIbinding is reduced, wherein induction of ADCP function is reduced butresidual ADCP function is preserved, compared to effector functioninduced by rituximab, and wherein induction of direct cell death isincreased compared to direct cell death induced by rituximab.

Antibodies according to the present invention comprising amino acidmodifications (substitutions, additions, deletions) can be prepared bymethods known in the art. These methods include, but are not limited to,preparation by site-directed (or oligonucleotide-mediated) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared nucleicacid encoding the polypeptide. Site-directed mutagenesis is a preferredmethod for preparing substitution variants. This technique is well knownin the art (see e.g., Carter eta Nucleic Acids Res. 13: 4431-4443 (1985)and Kunkel et. al, Proc. Natl. Acad. ScL USA 82: 488 (1987), each ofwhich is hereby incorporated by reference in its entirety). Briefly, incarrying out site directed mutagenesis of DNA, the starting DNA isaltered by first hybridizing an oligonucleotide encoding the desiredmutation to a single strand of such starting DNA. After hybridization, aDNA polymerase is used to synthesize an entire second strand, using thehybridized oligonucleotide as a primer, and using the single strand ofthe starting DNA as a template. Thus, the oligonucleotide encoding thedesired mutation is incorporated in the resulting double-stranded DNA.

PCR mutagenesis is also suitable for making amino acid sequence variantsof the non-modified starting polypeptide (see, e.g., Vallette et. al,Nuc. Acids Res. 17: 723-733 (1989), hereby incorporated by reference inits entirety). Briefly, when small amounts of template DNA are used asstarting material in a PCR, primers that differ slightly in sequencefrom the corresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template.

Another method according to the invention for preparing the inventiveantibody variants, cassette mutagenesis, is based on the techniquedescribed by Wells et al, Gene 34: 315-323 (1985), hereby incorporatedby reference in its entirety. The starting material is the plasmid (orother vector) comprising the starting polypeptide DNA to be modified.The codon(s) in the starting DNA to be mutated are identified. Theremust be a unique restriction endonuclease site on each side of theidentified mutation site(s). If no such restriction sites exist, theycan be generated using the herein-disclosed oligonucleotide-mediatedmutagenesis method to introduce them at appropriate locations in thestarting polypeptide DNA. The plasmid DNA is cut at these sites tolinearize it. A double-stranded oligonucleotide encoding the sequence ofthe DNA between the restriction sites but containing the desiredmutation(s) is synthesized using standard procedures, wherein the twostrands of the oligonucleotide are synthesized separately and thenhybridized together using standard techniques. This double-strandedoligonucleotide is referred to as the cassette. This cassette isdesigned to have 5′ and 3′ ends that are compatible with the ends of thelinearized plasmid, such that it can be directly ligated to the plasmid.This plasmid now contains the mutated DNA sequence.

Alternatively, or additionally, the desired amino acid sequence encodingan antibody variant can be determined, and a nucleic acid sequenceencoding such an amino acid sequence variant can be generatedsynthetically.

By introducing the appropriate amino acid sequence modifications in aparent Fc region, one can generate a variant Fc region which (a)mediates one or more effector functions in the presence of humaneffector cells less effectively and/or (b) binds an Fcγ receptor (FcγR)with smaller affinity than the parent polypeptide. Such modified Fcregions will comprise at least one amino acid modification in the Fcregion. Likewise, variant CH1 and VH regions mediating altered inductionof direct cell death more or less effectively compared to the parentnon-substituted polypeptide can be generated by introducing theappropriate amino acid sequence modifications. Combined Fc and CH1and/or VH region variants can be generated by introducing substitutionseither individually into parent region polypeptides and combiningmodified regions to a combined Fc and CH1 and/or VH variant or byintroducing all amino acid substitutions at the same time.

In preferred embodiments, the parent polypeptide Fc region is a human Fcregion, including but not limited to a native human Fc region human IgG1(A and non-A allotypes), IgG2, IgG3, IgG4, and all allotypes known ordiscovered from any species Fc region. Such regions have sequences suchas those disclosed in U.S. Provisional Patent Application No.60/678,776, which is hereby incorporated by reference in its entirety.

In certain embodiments, in order to generate an antibody comprising oneor more amino acid substitutions in the heavy chain CH1 and VH regionsfurther comprising a modified Fc region with altered effector function(including but not limited to ADCC, CDC and ADCP), the parentpolypeptide preferably has pre-existing effector function (e.g., theparent polypeptide comprises a human IgG1 or human IgG3 Fc region). Insome embodiments, a modified Fc region with altered effector functionmediates effector function (including but not limited to ADCC, CDC andADCP) substantially less effectively than an antibody with a nativesequence IgG1 or IgG3 Fc region.

The polypeptides of the invention having modified heavy chain regionscan be subjected to one or more further modifications, depending on thedesired or intended use of the polypeptide. Such modifications involve,for example but are not limited to, further alteration of the amino acidsequence (substitution, insertion and/or deletion of amino acidresidues), fusion to heterologous polypeptide(s) and/or covalentmodifications. Such further modifications can be made prior to,simultaneously with, or following, the amino acid modification(s)disclosed herein which result in an alteration of signaling activityand/or of Fc receptor binding and/or effector function.

In another aspect of the invention, an antibody provided herein has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM. Preferably, the dissociation constant is 10⁻⁸ Mor less, from 10⁻⁸ M to 10⁻¹³ M, from 10⁻⁹ M to 10⁻¹³ M.

In one aspect of the invention, the dissociation constant Kd is measuredby scatchard analysis using Europium labeled antibodies and analyzingthe bound/free ratio at different antibody concentrations. 2×10⁵ SU-DHL4cells are seeded into V-bottom plates (NUNC) in culture mediumcontaining 20% FCS (50 μl). Then, 50 μl Europium-labeled antibodies areadded in different concentrations and incubated at 25° C. for 1 h.Thereafter, 150 μl complete medium is added and the plate iscentrifuged. The cells are washed 2× by replacing the whole medium,transferred into a new plate and washed again 2×. Finally, the medium isremoved after centrifugation and the pellet is resuspended in 200 μlenhancer solution, transferred into a black 96 well plate and put onto ashaker for 10 min. By this step, the coupled Europium is released intothe supernatant, the fluorescence is enhanced and then analyzed on a BMGPheraStar machine (ex337/em615). The molarity of the antibodies bound iscalculated by usage of relative fluorescence units (RFU) from a standard(Europium-antibody) titration curve. The amount of free antibody iscalculated by subtraction of the bound antibody from the signal measuredin the total antibody wells. The bound versus free antibody ratio isplotted against the number of bound antibody molecules and the slope ofthe curve (S) is determined. The affinity of the antibody is determinedusing the following formula: Kd (M)=1/−S.

According to another embodiment, the dissociation constant Kd ismeasured by a radiolabeled antigen or Fc receptor binding assay (RIA)performed with the Fab version of an antibody of interest and itsantigen as disclosed by the following assay. Solution binding affinityof Fabs for antigen is measured by equilibrating Fab with a minimalconcentration of (¹²⁵I)-labeled antigen in the presence of a titrationseries of unlabeled antigen, then capturing bound antigen with ananti-Fab antibody-coated plate (see, e.g., Chen, et al., J. Mol. Biol.293 (1999) 865-881). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Sigma-Aldrich P7366), 100 pM or 26 pM[¹²⁵I]-antigen are mixed with serial dilutions of a Fab of interest(e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, inPresta, et al., Cancer Res. 57 (1997) 4593-4599). The Fab of interest isthen incubated overnight; however, the incubation may continue for alonger period (e.g., about 65 hours) to ensure that equilibrium isreached. Thereafter, the mixtures are transferred to the capture platefor incubation at room temperature for one hour. The solution is thenremoved and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, the dissociation constant Kd of theantibody is measured using surface plasmon resonance assays using aBIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at25° C. with immobilized antigen or Fc receptor CMS chips at −10 responseunits (RU). Briefly, carboxy methylated dextran biosensor chips (CMS,BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (−0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (kon) and dissociation rates (koff) arecalculated using a simple one-to-one Langmuir binding model (BIACORE®Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensograms. Kd is calculated as the ratiokoff/kon. See, e.g., Chen, et al., J. Mol. Biol. 293 (1999) 865-881. Ifthe on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assayherein, then the on-rate can be determined by using a fluorescentquenching technique that measures the increase or decrease influorescence emission intensity (excitation=295 nm; emission=340 nm, 16nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) inPBS, pH 7.2, in the presence of increasing concentrations of antigen asmeasured in a spectrometer, such as a stop-flow equipped spectrophometer(Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

In a specific embodiment, the parent non-substituted heavy chain regionis from obinutuzumab, as disclosed in SEQ ID NO: 1, and furtherdisclosed herein. The amino acid positions 11, 151, and 297 according toKabat are underlined.

Obinutuzumab heavy chain amino acid sequence  (SEQ ID NO: 1) QVQLVQSGAEV KKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY N STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In another specific embodiment, the parent non-substituted heavy chainregion is from rituximab, as disclosed in SEQ ID NO: 2, and furtherdisclosed herein. The amino acid positions 11, 151, and 297 according toKabat are underlined.

Rituximab heavy chain amino acid sequence  (SEQ ID NO: 2) QVQLQQPGAE LVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Expression of Modified Antibodies

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of amodified antibody having substantially the same binding specificity of aparent antibody along with appropriate transcriptional/translationalcontrol signals. These methods include in vitro recombinant DNAtechniques, synthetic techniques and in vivo recombination/geneticrecombination. See, for example, the techniques described in Maniatis etal, MOLECULAR CLONING A LABORATORY MANUAL, Cold Spring HarborLaboratory, N. Y. (1989) and Ausubel et at, CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience,N.Y (1989).

A variety of host-expression vector systems can be utilized to expressthe coding sequence of the antibodies of the present invention.Preferably, mammalian cells are used as host cell systems transfectedwith recombinant plasmid DNA or cosmid DNA expression vectors containingthe coding sequence of the protein of interest and the coding sequenceof the fusion polypeptide. Most preferably, CHO cells, HEK293-EBNAcells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, othermammalian cells, yeast cells, insect cells, or plant cells are used ashost cell system. Some examples of expression systems and selectionmethods are disclosed in the following references, and referencestherein: Borth et at, Biotechnol. Bioen. 71(4):266-73 (2000-2001), inWerner et al, Arzneimittelforschung/Drug Res. 48(8):870-80 (1998), inAndersen and Krummen, Curr. Op. Biotechnol. 13:117-123 (2002), in Chaddand Chamow, Curr. Op. Biotechnol. 12:188-194 (2001), and in Giddings,Curr. Op. Biotechnol. 12: 450-454 (2001). In alternate embodiments,other eukaryotic host cell systems may be contemplated, including yeastcells transformed with recombinant yeast expression vectors containingthe coding sequence of an antibody of the present invention, such as theexpression systems taught in U.S. Pat. Appl. No. 60/344,169 and WO03/056914 (methods for producing human-like glycoprotein in a non-humaneukaryotic host cell) (each of which is hereby incorporated by referencein its entirety); insect cell systems infected with recombinant virusexpression vectors (e.g., baculo virus) containing the coding sequenceof a modified antibody having substantially the same binding specificityof a parent antibody; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing the coding sequence of the antibody of theinvention, including, but not limited to, the expression systems taughtin U.S. Pat. No. 6,815,184 (methods for expression and secretion ofbiologically active polypeptides from genetically engineered duckweed);WO 2004/057002 (production of glycosylated proteins in bryophyte plantcells by introduction of a glycosyl transferase gene) and WO 2004/024927(methods of generating extracellular heterologous non-plant protein inmoss protoplast); and U.S. Pat. Appl. Nos. 60/365,769, 60/368,047, andWO 2003/078614 (glycoprotein processing in transgenic plants comprisinga functional mammalian GnTIII enzyme) (each of which is herebyincorporated by reference in its entirety); or animal cell systemsinfected with recombinant virus expression vectors (e.g., adenovirus,vaccinia virus) including cell lines engineered to contain multiplecopies of the DNA encoding a modified antibody having substantially thesame binding specificity of a parent antibody either stably amplified(CHO/dhfr) or unstably amplified in double-minute chromosomes (e.g.,murine cell lines). In one embodiment, the vector comprising thepolynucleotide(s) encoding the antibody of the invention ispolycistronic. In a preferred embodiment, the antibody is a humanizedantibody.

In one embodiment, the present invention is directed to an expressionvector and/or a host cell which comprise one or more isolatedpolynucleotides of the present invention. According to one aspect of theinvention, the light and heavy chains can be expressed separately, usingimmunoglobulin light chain and immunoglobulin heavy chains in separateplasmids, or on a single (including but not limited to, a polycistronic)vector. Accordingly, in one aspect of the invention, a polynucleotideencoding a variant heavy chain region of an antibody is provided. In oneaspect of the invention, a polynucleotide encoding a light chain regionof an antibody is provided. In one aspect of the invention a vectorcomprising at least one polynucleotide encoding a variant heavy chainand/or a light chain of an antibody is provided. In a further aspectsaid vector is polycystronic. One embodiment of the present invention isdirected to host cells comprising said polynucleotides or vectors. Thepresent invention is also directed to a method for producing an antibodyof the present invention in a host cell comprising (i) culturing thehost cell under conditions permitting the expression of said at leastone polynucleotide; and (ii) recovering said antibody from the culturemedium.

For the methods of this invention, stable expression is generallypreferred to transient expression because it typically achieves morereproducible results and also is more amenable to large-scaleproduction. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with therespective coding nucleic acids controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows selection of cells whichhave stably integrated the plasmid into their chromosomes and grow toform foci which in turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al, Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sd. USA 48:2026 (1962)), and adeninephosphoribosyltransferase (Lowy et al, Cell 22:817 (1980)) genes, whichcan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al, Natl Acad.Sd. USA 77:3567 (1989); O'Hare et al, Proc. Natl Acad. ScL USA 78: 1527(1981)); gpt, which confers resistance to mycophenolic acid (Mulligan &Berg, Proc. Natl Acad. ScL USA 78:2072 (1981)); neo, which confersresistance to the aminoglycoside G-418 (Colberre-Garapin et al, J. MoIBiol 150:1 (1981)); and hygro, which confers resistance to hygromycin(Santerre et al, Gene 30:147 (1984) genes. Recently, additionalselectable genes have been described, namely trpB, which allows cells toutilize indole in place of tryptophan; hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. NatlAcad. ScL USA S5:8047 (1988)); the glutamine synthase system; and ODC(ornithine decarboxylase) which confers resistance to the ornithinedecarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DEMO(McConlogue, in: Current Communications in Molecular Biology, ColdSpring Harbor Laboratory ed. (1987)).

Therapeutic Applications of Modified Antibodies According to the Methodsof the Invention

In the broadest sense, the modified antibodies of the present inventioncan be used to target cells in vivo or in vitro that express a targetantigen, in particular, where said target antigen is expressed on thecell surface. The cells expressing a target antigen can be targeted fordiagnostic or therapeutic purposes. In one aspect, the modifiedantibodies of the present invention can be used to control effectorfunction while altering cell signaling activity in cells expressing atarget antigen. In another aspect, the modified antibodies of thepresent invention can be used to suppress effector function and/or alterthe cross-linking and/or oligomerization of one or more target antigens.Target antigens for the modified antibodies of the present invention canbe cell surface receptors including, but not limited to CD20, CD21,CD22, CD19, CD47, CD99, CD2, CD45, Her1 (EGFR), Her2/neu, Her3, Her4,TRAIL receptors (e.g., TRAILR1, TRAILR2), TNFR, FGF receptors (e.g.,FGFR1), IGF receptors, PDGF receptors, VEGF receptors, and othercell-surface associated receptors. In a particular embodiment, thetarget antigen is CD20. The modified antibodies of the invention alsoact to arrest the cell cycle, cause direct cell death of the targetcells, inhibit angiogenesis and/or cause differentiation of targetcells.

In another aspect, the invention is directed to a method for treating adisease that is treatable by suppressing effector function and/or altercell signaling activity of a target antigen comprising administering atherapeutically effective amount of a modified antibody of the presentinvention to a subject in need thereof. In a specific embodiment themodified antibody is humanized. Examples of diseases for which themodified antibodies can be administered include, but are not limited to,cell proliferation diseases or disorders, autoimmune diseases ordisorders, and diseases or disorders related to bacterial or viralinfection.

In one embodiment, the invention is directed to a method for treating adisease selected from the group consisting of proliferative disorder andautoimmune disease comprising administering to an individual aneffective amount of the antibody according to the present invention. Ina further aspect, said method comprises administering to a subject apharmaceutically effective amount of a composition containing at leastone of the modified antibodies of the invention (conjugated, includingbut not limited to an immunotoxin, or unconjugated). In one embodiment,said proliferative disorder include, but is not limited to, neoplasms,cancers, malignancies and/or tumors located in the abdomen, bone,breast, digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvic, skin, soft tissue, spleen, thoracic region, andurogenital system. Particular neoplasms, cancers, malignancies, and/ortumors that can be treated with the antibodies of the invention include,but are not limited to, epidermal and squamous cell carcinomas, gliomas,pancreatic cancer, ovarian cancer, prostate cancer, breast cancer,bladder cancer, head and neck cancer, renal cell carcinomas, coloncancer, colorectal cancer, lung cancer, brain tumor, malignant melanoma,leukemia, lymphomas, T cell lymphomas, multiple myeloma, gastric cancer,cervical cancer, endometrial carcinoma, esophageal cancer, liver cancer,cutaneous cancer, urinary tract carcinoma, choriocarcinoma, pharyngealcancer, laryngeal cancer, thecomatosis, androblastoma, endometriumhyperplasy, endometriosis, embryoma, fibrosarcoma, Kaposi's sarcoma,hemangioma, cavernous hemangioma, angioblastoma, retinoblastoma,astrocytoma, neurofibroma, oligodendroglioma, medulloblastoma,ganglioneuroblastoma, glioma, rhabdomyosarcoma, hamartoblastoma,osteogenic sarcoma, leiomyosarcoma, thyroid sarcoma, Ewing's sarcoma,and Wilms tumor. In a preferred embodiment, said proliferative disorderis a CD20 expressing cancer. In another preferred embodiment, saidcancer is selected from the group consisting of lymphoma and lymphocyticleukemia. Such lymphomas and lymphocytic leukemias include, but are notlimited to, follicular lymphomas, Small Non-Cleaved CellLymphomas/Burkitt's lymphoma (including endemic Burkitt's lymphoma,sporadic Burkitt's lymphoma and Non-Burkitt's lymphoma), marginal zonelymphomas (including extranodal marginal zone B cell lymphoma(Mucosa-associated lymphatic tissue lymphomas, MALT), nodal marginalzone B cell lymphoma and splenic marginal zone lymphoma), Mantle celllymphoma (MCL), Large Cell Lymphoma (including diffuse large B-celllymphoma (DLBCL), Diffuse Mixed Cell Lymphoma, Immunoblastic Lymphoma,Primary Mediastinal B-Cell Lymphoma, Angiocentric Lymphoma-PulmonaryB-Cell Lymphoma), hairy cell leukemia, lymphocytic lymphoma,Waldenstrom's macroglobulinemia, acute lymphocytic leukemia (ALL),chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL),B-cell prolymphocytic leukemia, plasma cell neoplasms, plasma cellmyeloma, multiple myeloma, plasmacytoma, Hodgkin's disease. In apreferred embodiment, said CD20 expressing cancer is selected from thegroup consisting of Non-Hodgkin's lymphomas (NHL), follicular lymphomas,diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia(CLL).

In a further aspect, the invention is directed to an improved method fortreating B-cell proliferative disorders including B-cell lymphoma, basedon B-cell depletion comprising administering a therapeutically effectiveamount of an antibody of the present invention to a human subject inneed thereof. In a preferred embodiment, the antibody is an anti-CD20antibody with a binding specificity substantially the same as that ofthe murine B-Ly1 antibody. In another preferred embodiment the antibodyis humanized. In another preferred embodiment the antibody comprises avariant heavy chain region comprising at least one amino acidsubstitution relative to the parent non-substituted heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residues Pro151 and Asn297, wherein saidsubstitutions are Pro151Phe and Asn297Asp, wherein induction of effectorfunction is reduced or abolished, and wherein direct cell death inducedby the antibody comprising the variant heavy chain region is increasedcompared to effector function and direct cell death induced by theantibody comprising the parent non-substituted heavy chain region. Inthis aspect of the invention, the antibodies of the invention are usedto deplete the blood of normal B-cells for an extended period.

The subject invention further provides methods for inhibiting the growthof human tumor cells, treating a tumor in a subject, and treating aproliferative type disease in a subject. These methods compriseadministering to the subject an effective amount of the composition ofthe invention.

Other cell proliferation disorders can also be treated with the modifiedantibodies of the present invention. As encompassed by the presentinvention, said cell proliferation disorders include, but are notlimited to hypergammaglobulinemia, lymphoproliferative disorders,paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstrom'sMacroglobulinemia, Gaucher's Disease, histiocytosis, and any other cellproliferation disease, besides neoplasia, located in an organ systemlisted herein.

In another embodiment, the invention is directed to a method fortreating an autoimmune disease. In one embodiment, said autoimmunedisease include, but is not limited to, immune-mediatedthrombocytopenias, such as acute idiopathic thrombocytopenic purpureaand chronic idiopathic thrombocytopenic purpurea, dermatomyositis,Sydenham's chorea, lupus nephritis, rheumatic fever, polyglandularsyndromes, Henoch-Schonlein purpura, poststreptococcal nephritis,erythema nodosum, Takayasu's arteritis, Addison's disease, erythemamultiforme, polyarteritis nodosa, ankylosing spondylitis, Goodpasture'ssyndrome, thromboangitis ubiterans, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, polymyaglia, pernicious anemia, rapidlyprogressive glomerulonephritis and fibrosing alveolitis, inflammatoryresponses such as inflammatory skin diseases including psoriasis anddermatitis (e.g., atopic dermatitis), systemic scleroderma andsclerosis, responses associated with inflammatory bowel disease (such asCrohn's disease and ulcerative colitis), respiratory distress syndrome(including adult respiratory distress syndrome, ARDS), dermatitis,meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergicconditions such as eczema and asthma and other conditions involvinginfiltration of T cells and chronic inflammatory responses,atherosclerosis, leukocyte adhesion deficiency, rheumatoid arthritis,systemic lupus erythematosus (SLE), diabetes mellitus (e.g., Type 1diabetes mellitus or insulin dependent diabetes mellitus), multiplesclerosis, Reynaud's syndrome, autoimmune thyroiditis, allergicencephalomyelitis, Sjögren's syndrome, juvenile onset diabetes, andimmune responses associated with acute and delayed hypersensitivitymediated by cytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, polymyositis, granulomatosis and vasculitis, perniciousamenia (Addison's disease), diseases involving leukocyte diapedesis,central nervous system (CNS) inflammatory disorder, multiple organinjury syndrome, hemolytic anemia (including, but not limited tocryoglobinemia or Coombs positive anemia), myasthenia gravis,antigen-antibody complex mediated diseases, anti-glomerular basementmembrane disease, antiphospholipid syndrome, allergic neuritis, Graves'disease, Lambert-Eaton myasthenic syndrome, pemphigoid bullous,pemphigus, autoimmune polyendocrinopathies, Reiter's disease, stiff-mansyndrome, Behcet disease, giant cell arteritis, immune complexnephritis, IgA nephropathy, IgM polyneuropathies, immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia. In apreferred embodiment, said autoimmune disease is selected from the groupconsisting of rheumatoid arthritis, lupus, multiple sclerosis, Sjögren'ssyndrome and transplant rejection.

In a further aspect, the invention is directed to an improved method fortreating an autoimmune disease as defined herein, based on B-celldepletion comprising administering a therapeutically effective amount ofan antibody of the present invention to a human subject in need thereof.In a preferred embodiment, the antibody is a anti-CD20 antibody with abinding specificity substantially the same as that of the murine B-Ly1antibody. In another preferred embodiment the antibody is humanized. Inanother preferred embodiment the antibody comprises a variant heavychain region comprising at least one amino acid substitution relative tothe parent non-substituted heavy chain region, wherein the heavy chainregion of the parent non-substituted antibody comprises the amino acidresidues Pro151 and Asn297, wherein said substitutions are Pro151Phe andAsn297Asp, wherein induction of effector function is reduced orabolished, and wherein direct cell death induced by the antibodycomprising the variant heavy chain region is increased compared toeffector function and direct cell death induced by the antibodycomprising the parent non-substituted heavy chain region. In this aspectof the invention, the antibodies of the invention are used to depletethe blood of normal B-cells for an extended period.

The modified antibodies of the present invention can be used alone or incombination with other treatments or therapeutic agents to treatdisorders that are treatable by altering effector function and/orincreasing or decreasing cell signaling activity and/or cross-linking ofone or more target antigens. In one embodiment, modified antibodies ofthe present invention can be used alone to target and kill tumor cellsin vivo. The modified antibodies can also be used in conjunction with anappropriate therapeutic agent to treat human carcinoma. For example, themodified antibodies can be used in combination with standard orconventional treatment methods such as chemotherapy, radiation therapyor can be conjugated or linked to a therapeutic drug, or toxin, as wellas to a lymphokine or a tumor-inhibitory growth factor, for delivery ofthe therapeutic agent to the site of the carcinoma. In particularembodiments, the conjugates of the modified antibodies of this inventioninclude (1) immunotoxins (conjugates of the modified antibody and acytotoxic moiety) and (2) labeled (e.g., radiolabeled, enzyme-labeled,or fluorochrome-labeled) modified antibodies in which the label providesa means for identifying immune complexes that include the labeledantibody. The cytotoxic moiety of the immunotoxin may be a cytotoxicdrug or an enzymatically active toxin of bacterial or plant origin, oran enzymatically active fragment (“A chain”) of such a toxin.Enzymatically active toxins and fragments thereof used are diphtheria Achain, nonbinding active fragments of diphtheria toxin, exotoxin A chain(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin Achain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordicacharantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor,gelonin, mitogellin, restrictocin, phenomycin, and enomycin. In anotherembodiment, the modified antibodies are conjugated to small moleculeanticancer drugs. Conjugates of the modified antibody and such cytotoxicmoieties are made using a variety of bifunctional protein couplingagents. Examples of such reagents are SPDP, IT, bifunctional derivativesof imidoesters such a dimethyl adipimidate HCl, active esters such asdisuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azidocompounds such as bis (p-azidobenzoyl) hexanediamine, bis-diazoniumderivatives such as bis-(p-diazoniumbenzoyl)-ethylenediamine,diisocyanates such as tolylene 2,6-diisocyanate, and bis-active fluorinecompounds such as 1,5-difluoro-2,4-dinitrobenzene. The lysing portion ofa toxin may be joined to the Fab fragment of the modified antibodies.Additional appropriate toxins are known in the art, as evidenced ine.g., published U.S. Patent Application No. 2002/0128448, incorporatedherein by reference in its entirety.

In one embodiment, the antigen binding molecule of the present inventionis conjugated to an additional moiety, such as a radiolabel or a toxin.Such conjugated modified antibodies can be produced by numerous methodsthat are well known in the art.

A variety of radionuclides are applicable to the present invention andthose skilled in the art are credited with the ability to readilydetermine which radionuclide is most appropriate under a variety ofcircumstances. For example, ¹³¹iodine is a well known radionuclide usedfor targeted immunotherapy. However, the clinical usefulness of¹³¹iodine can be limited by several factors including: eight-dayphysical half-life; dehalogenation of iodinated antibody both in theblood and at tumor sites; and emission characteristics (eg, large gammacomponent) which can be suboptimal for localized dose deposition intumor. With the advent of superior chelating agents, the opportunity forattaching metal chelating groups to proteins has increased theopportunities to utilize other radionuclides such as ¹¹¹indium and⁹⁰yttrium. ⁹⁰Yttrium provides several benefits for utilization inradioimmunotherapeutic applications: the 64 hour half-life of ⁹⁰yttriumis long enough to allow antibody accumulation by tumor and, unlike eg,¹³¹iodine, ⁹⁰yttrium is a pure beta emitter of high energy with noaccompanying gamma irradiation in its decay, with a range in tissue of100 to 1000 cell diameters. Furthermore, the minimal amount ofpenetrating radiation allows for outpatient administration of⁹⁰yttrium-labeled antibodies. Additionally, internalization of labeledantibody is not required for cell killing, and the local emission ofionizing radiation should be lethal for adjacent tumor cells lacking thetarget antigen.

Effective single treatment dosages (i.e., therapeutically effectiveamounts) of ⁹⁰yttrium labeled modified antibodies of the presentinvention range from between about 5 and about 75 mCi, more preferablybetween about 10 and about 40 mCi. Effective single treatment non-marrowablative dosages of ¹³iodine labeled antibodies of the present inventionrange from between about 5 and about 70 mCi, more preferably betweenabout 5 and about 40 mCi. Effective single treatment ablative dosages(i.e., may require autologous bone marrow transplantation) of ¹³¹iodinelabeled antibodies of the present invention range from between about 30and about 600 mCi, more preferably between about 50 and less than about500 mCi. In conjunction with a chimeric antibody according to thepresent invention, owing to the longer circulating half life vis-a-vismurine antibodies, an effective single treatment non-marrow ablativedosages of ¹³¹iodine labeled chimeric antibodies range from betweenabout 5 and about 40 mCi, more preferably less than about 30 mCi.Imaging criteria for the ¹¹¹indium label, are typically less than about5 mCi.

With respect to radiolabeled antibodies of the present invention,therapy therewith can also occur using a single therapy treatment orusing multiple treatments. Because of the radionuclide component, it ispreferred that prior to treatment, peripheral stem cells (“PSC”) or bonemarrow (“BM”) be “harvested” for patients experiencing potentially fatalbone marrow toxicity resulting from radiation. BM and/or PSC areharvested using standard techniques, and then purged and frozen forpossible reinfusion. Additionally, it is most preferred that prior totreatment a diagnostic dosimetry study using a diagnostic labeledantibody (including but not limited to using ¹¹¹indium) be conducted onthe patient, a purpose of which is to ensure that the therapeuticallylabeled antibody (eg, using ⁹⁰yttrium) will not become unnecessarily“concentrated” in any normal organ or tissue.

In one embodiment, a chimeric, modified antibody of the presentinvention, is conjugated to ricin A chain. Most advantageously, thericin A chain is deglycosylated and produced through recombinant means.An advantageous method of making the ricin immunotoxin is described inVitetta et al., Science 238, 1098 (1987), hereby incorporated byreference in its entirety.

When used to kill human cancer cells in vitro for diagnostic purposes,the conjugates will typically be added to the cell culture medium at aconcentration of at least about 10 nM. The formulation and mode ofadministration for in vitro use are not critical. Aqueous formulationsthat are compatible with the culture or perfusion medium will normallybe used. Cytotoxicity may be read by conventional techniques todetermine the presence or degree of cancer.

As discussed herein, a cytotoxic radiopharmaceutical for treating cancermay be made by conjugating a radioactive isotope (e.g., I, Y, Pr) to achimeric, modified antibody of the present invention. The term“cytotoxic moiety” as used herein is intended to include such isotopes.

In another embodiment, liposomes are filled with a cytotoxic drug andthe liposomes are coated with the antibodies of the present invention.Because many of the target molecules for the modified antibodies of thepresent invention are expressed on the cell surface (e.g., there aremany CD20 molecules on the surface of the malignant B-cell), this methodpermits delivery of large amounts of drug to the correct cell type.

Techniques for conjugating such therapeutic agents to antibodies arewell known (see, e.g., Amon et al., “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy”, in Monoclonal Antibodiesand Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et at, “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson etal. (eds.), pp. 623-53(Marcel Defcker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982) (each of which is hereby incorporated by reference in itsentirety).

Still other therapeutic applications for the antibodies of the inventioninclude conjugation or linkage, including but not limited to conjugationby recombinant DNA techniques, to an enzyme capable of converting aprodrug into a cytotoxic drug and the use of that antibody-enzymeconjugate in combination with the prodrug to convert the prodrug to acytotoxic agent at the tumor site (see, e.g., Senter et al., “Anti-TumorEffects of Antibody-alkaline Phosphatase”, Proc. Natl. Acad. ScL USA55:4842-46 (1988); “Enhancement of the in vitro and in vivo AntitumorActivites of Phosphorylated Mitocycin C and Etoposide Derivatives byMonoclonal Antibody-Alkaline Phosphatase Conjugates”, Cancer Research49:5789-5792 (1989); and Senter, “Activation of Prodrugs byAntibody-Enzyme Conjugates: A New Approach to Cancer Therapy,” FASEB J.4:188-193 (1990)).

Still another therapeutic use for the antibodies of the inventioninvolves use, either unconjugated, or as part of an antibody-drug orantibody-toxin conjugate, to remove tumor cells from the bone marrow ofcancer patients. According to this approach, autologous bone marrow maybe purged ex vivo by treatment with the antibody and the marrow infusedback into the patient (see, e.g., Ramsay et al., “Bone Marrow PurgingUsing Monoclonal Antibodies”, J. Clin. Immunol, 8(2):81-88 (1988)).

Furthermore, it is contemplated that the invention comprises asingle-chain immunotoxin comprising antigen binding domains that allowsubstantially the same specificity of binding as a parent antibody(including but not limited to, polypeptides comprising the CDRs of theparent antibody) and further comprising a toxin polypeptide. Thesingle-chain immunotoxins of the invention may be used to treat humancarcinoma in vivo.

Similarly, a fusion protein comprising at least the antigen-bindingregion of an antibody of the invention joined to at least a functionallyactive portion of a second protein having anti-tumor activity, includingbut not limited to, a lymphokine or oncostatin, can be used to treathuman carcinoma in vivo.

Accordingly, the present invention provides a method for selectivelykilling tumor cells expressing cell surface receptors including, but notlimited to CD20, Her1 (EGFR), Her2/neu, Her3, Her4, TRAIL receptors(e.g., TRAILR1, TRAILR2), TNFR, FGF receptors (e.g., FGFR1), IGFreceptors, PDGF receptors, VEGF receptors, and other cell-surfaceassociated receptors. This method comprises reacting the modifiedantibody of the invention (conjugated, e.g., as an immunotoxin, orunconjugated) with said tumor cells. These tumor cells maybe from ahuman carcinoma.

In a further aspect, the invention relates to an antibody according tothe present invention for use as a medicament. In one embodiment, theinvention relates to an antibody according to the present invention foruse in treating a disease selected from the group consisting ofproliferative disorder and autoimmune disease. According to one aspectof the invention said proliferative disorder is selected from the groupconsisting of B-cell lymphoma, lung cancer, non-small cell lung (NSCL)cancer, bronchioalviolar cell lung cancer, bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,chronic or acute leukemia, lymphocytic lymphomas, neoplasms of thecentral nervous system (CNS), spinal axis tumors, brain stem glioma,glioblastoma multiforme, astrocytomas, schwannomas, ependymomas,medulloblastomas, meningiomas, squamous cell carcinomas, pituitaryadenomas, including refractory versions of any of the herein cancers, ora combination of one or more of the herein cancers or a precancerouscondition or lesion herein disclosed. The precancerous condition orlesion includes, for example, the group consisting of oral leukoplakia,actinic keratosis (solar keratosis), precancerous polyps of the colon orrectum, gastric epithelial dysplasia, adenomatous dysplasia, hereditarynonpolyposis colon cancer syndrome (HNPCC), Barrett's esophagus, bladderdysplasia, and precancerous cervical conditions. Preferably, the canceris selected from the group consisting of B-cell lymphoma, breast cancer,bladder cancer, head and neck cancer, skin cancer, pancreatic cancer,lung cancer, ovarian cancer, colon cancer, prostate cancer, kidneycancer, and brain cancer. In a preferred embodiment, said proliferativedisorder is a CD20 expressing cancer. In another preferred embodiment,said cancer is selected from the group consisting of lymphoma andlymphocytic leukemia.

Accordingly, in one aspect of the invention said autoimmune disease isselected from the group consisting of immune-mediated thrombocytopenias,such as acute idiopathic thrombocytopenic purpurea and chronicidiopathic thrombocytopenic purpurea, dermatomyositis, Sydenham'schorea, lupus nephritis, rheumatic fever, polyglandular syndromes,Henoch-Schonlein purpura, poststreptococcal nephritis, erythema nodosum,Takayasu's arteritis, Addison's disease, erythema multiforme,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis ubiterans, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, polymyaglia, pernicious anemia, rapidlyprogressive glomerulonephritis and fibrosing alveolitis, inflammatoryresponses such as inflammatory skin diseases including psoriasis anddermatitis (e.g., atopic dermatitis), systemic scleroderma andsclerosis, responses associated with inflammatory bowel disease (such asCrohn's disease and ulcerative colitis), respiratory distress syndrome(including adult respiratory distress syndrome, ARDS), dermatitis,meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergicconditions such as eczema and asthma and other conditions involvinginfiltration of T cells and chronic inflammatory responses,atherosclerosis, leukocyte adhesion deficiency, rheumatoid arthritis,systemic lupus erythematosus (SLE), diabetes mellitus (e.g., Type 1diabetes mellitus or insulin dependent diabetes mellitus), multiplesclerosis, Reynaud's syndrome, autoimmune thyroiditis, allergicencephalomyelitis, Sjögren's syndrome, juvenile onset diabetes, andimmune responses associated with acute and delayed hypersensitivitymediated by cytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, polymyositis, granulomatosis and vasculitis, perniciousamenia (Addison's disease), diseases involving leukocyte diapedesis,central nervous system (CNS) inflammatory disorder, multiple organinjury syndrome, hemolytic anemia (including, but not limited tocryoglobinemia or Coombs positive anemia), myasthenia gravis,antigen-antibody complex mediated diseases, anti-glomerular basementmembrane disease, antiphospholipid syndrome, allergic neuritis, Graves'disease, Lambert-Eaton myasthenic syndrome, pemphigoid bullous,pemphigus, autoimmune polyendocrinopathies, Reiter's disease, stiff-mansyndrome, Behcet disease, giant cell arteritis, immune complexnephritis, IgA nephropathy, IgM polyneuropathies, immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia. In apreferred embodiment, said autoimmune disease is selected from the groupconsisting of rheumatoid arthritis, lupus, multiple sclerosis, Sjögren'ssyndrome and transplant rejection.

Yet another embodiment is the use of the antibody according to thepresent invention for the manufacture of a medicament for the treatmentor prophylaxis of cancer or for the treatment or prophylaxis of aprecancerous condition or lesion or for the treatment of an autoimmunedisease. Cancer and precancerous condition or lesions are defined asherein. In one embodiment, said cancer is a CD20 expressing cancer. In aspecific embodiment said cancer is a lymphoma or lymphocytic leukemia.In another specific embodiment said cancer is selected from the groupconsisting of follicular lymphomas, Small Non-Cleaved CellLymphomas/Burkitt's lymphoma (including endemic Burkitt's lymphoma,sporadic Burkitt's lymphoma and Non-Burkitt's lymphoma), marginal zonelymphomas (including extranodal marginal zone B cell lymphoma(Mucosa-associated lymphatic tissue lymphomas, MALT), nodal marginalzone B cell lymphoma and splenic marginal zone lymphoma), Mantle celllymphoma (MCL), Large Cell Lymphoma (including diffuse large B-celllymphoma (DLBCL), Diffuse Mixed Cell Lymphoma, Immunoblastic Lymphoma,Primary Mediastinal B-Cell Lymphoma, Angiocentric Lymphoma-PulmonaryB-Cell Lymphoma), hairy cell leukemia, lymphocytic lymphoma,Waldenstrom's macroglobulinemia, acute lymphocytic leukemia (ALL),chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL),B-cell prolymphocytic leukemia, plasma cell neoplasms, plasma cellmyeloma, multiple myeloma, plasmacytoma, Hodgkin's disease. In apreferred embodiment, said cancer is selected from the group consistingof Non-Hodgkin's lymphomas (NHL), follicular lymphomas, diffuse largeB-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In apreferred embodiment, said cancer is selected from the group consistingof lymphoma and lymphocytic leukemia. In another preferred embodiment,said autoimmune disease is selected from the group consisting ofrheumatoid arthritis, lupus, multiple sclerosis, Sjögren's syndrome andtransplant rejection.

The present invention encompasses pharmaceutical compositions,combinations, uses, and methods for treating human carcinomas andautoimmune diseases. The invention includes pharmaceutical compositionsfor use in the treatment of human carcinomas and autoimmune diseasescomprising a pharmaceutically effective amount of an antibody of thepresent invention and a pharmaceutically acceptable carrier.

The antibody compositions of the invention can be administered usingconventional modes of administration including, but not limited to,intravenous, intraperitoneal, oral, intralymphatic or administrationdirectly into the tumor. Intravenous administration is preferred.

The present invention is further directed to pharmaceutical compositionscomprising the modified antibodies of the present invention and apharmaceutically acceptable carrier. In one aspect of the invention,therapeutic formulations containing the antibodies of the invention areprepared for storage by mixing an antibody having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Pharmaceutically acceptable carriers are generally nontoxicto recipients at the dosages and concentrations employed, and include,but are not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG).

Lyophilized formulations adapted for subcutaneous administration aredescribed in WO 97/04801. Such lyophilized formulations may bereconstituted with a suitable diluent to a high protein concentrationand the reconstituted formulation may be administered subcutaneously tothe individual to be treated herein.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide a cytotoxic agent,chemotherapeutic agent, cytokine or immunosuppressive agent (e.g. onewhich acts on T cells, such as cyclosporin or an antibody that binds Tcells, e.g., one which binds LFA-1). The effective amount of such otheragents depends on the amount of antagonist present in the formulation,the type of disease or disorder or treatment, and other factorsdiscussed herein. These are generally used in the same dosages and withadministration routes as used hereinbefore or about from 1 to 99% of theheretofore employed dosages.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitablesustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antagonist, which matrices are inthe form of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andγ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The compositions of the invention may be in a variety of dosage formswhich include, but are not limited to, liquid solutions or suspension,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The compositions of the invention also preferably include conventionalpharmaceutically acceptable carriers and adjuvants known in the art suchas human serum albumin, ion exchangers, alumina, lecithin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,and salts or electrolytes such as protamine sulfate.

The most effective mode of administration and dosage regimen for thepharmaceutical compositions of this invention depends upon the severityand course of the disease, the patient's health and response totreatment and the judgment of the treating physician. Accordingly, thedosages of the compositions should be titrated to the individualpatient. Nevertheless, an effective dose of the compositions of thisinvention will generally be in the range of from about 0.01 to about2000 mg/kg.

The dosages of the present invention may, in some cases, be determinedby the use of predictive biomarkers. Predictive biomarkers are molecularmarkers that are used to determine (i.e., observe and/or quantitate) apattern of expression and/or activation of e.g., tumor related genes orproteins, or cellular components of a tumor-related signaling pathway.Elucidating the biological effects of targeted therapies in tumor tissueand correlating these effects with clinical response helps identify thepredominant growth and survival pathways operative in tumors, therebyestablishing a profile of likely responders and conversely providing arationale for designing strategies to overcoming resistance to therapy.

Predictive biomarkers may be measured by cellular assays that are wellknown in the art including, but not limited to immunohistochemistry,flow cytometry, immunofluorescence, capture-and-detection assays, andreversed phase assays, and/or assays set forth in U.S. Pat. Appl. Pub.No. 2004/0132097 A1, the entire contents of which is hereby incorporatedby reference in its entirety.

Thus, in one aspect, the present invention provides for a method fortreating a disorder that is related to altered or dysregulated cellsignaling by a target antigen and/or altered ability to mediatecross-linking and/or oligomerization of one or more target antigens,wherein reducing effector function mediated by the antibody therapy isbeneficial for the patient, comprising predicting a response to therapywith a modified antibody in a human subject in need of treatment byassaying a sample from the human subject prior to therapy with one or aplurality of reagents that detect expression and/or activation ofpredictive biomarkers for a disorder that is related to altered ordysregulated cell signaling by a target antigen and/or altered abilityto mediate cross-linking and/or oligomerization of one or more targetantigens (such as cancer); determining a pattern of expression and/oractivation of one or more of the predictive biomarkers, wherein thepattern predicts the human subject's response to the modified antibodytherapy; and administering to a human subject who is predicted torespond positively to modified antibody treatment a therapeuticallyeffective amount of a composition comprising a modified antibody of thepresent invention. As used herein, a human subject who is predicted torespond positively to modified antibody treatment is one for whom themodified antibody will have a measurable effect on the disease ordisorder that is related to altered or dysregulated cell signaling by atarget antigen and/or altered ability to mediate cross-linking and/oroligomerization of one or more target antigens (e.g., tumorregression/shrinkage) and for whom the benefits of modified antibodytherapy are not outweighed by adverse effects (e.g., toxicity). As usedherein, a sample means any biological sample from an organism,particularly a human, comprising one or more cells, including singlecells of any origin, tissue or biopsy samples which has been removedfrom organs such as breast, lung, gastrointestinal tract, skin, cervix,ovary, prostate, kidney, brain, head and neck, or any other organ ortissue of the body, and other body samples including, but not limitedto, smears, sputum, secretions, cerebrospinal fluid, bile, blood, lymphfluid, urine and feces.

The composition comprising a modified antibody of the present inventionwill be formulated, dosed, and administered in a fashion consistent withgood medical practice. Factors for consideration in this context includethe particular disease or disorder being treated, the particular mammalbeing treated, the clinic condition of the individual patient, the causeof the disease or disorder, the site of delivery of the agent, themethod of administration, the scheduling of administration, and otherfactors known to medical practitioners. The therapeutically effectiveamount of the antagonist to be administered will be governed by suchconsiderations.

In a preferred embodiment, the antibody modified according to thepresent invention is a humanized antibody. Suitable dosages for such anunconjugated antibody are, for example, in the range from about 20 mg/m²to about 1000 mg/m². In one embodiment, the dosage of the antibodymodified according to the present invention is equal to the dosagepresently recommended for the non-substituted parent antibody. In oneembodiment, the dosage of the antibody modified according to the presentinvention differs from the dosage presently recommended for thenon-substituted parent antibody. In one embodiment, the dosage of theantibody modified according to the present invention is lower comparedto the dosage presently recommended for the non-substituted parentantibody. In one embodiment, the dosage of the antibody modifiedaccording to the present invention is higher compared to the dosagepresently recommended for the non-substituted parent antibody. In oneembodiment, antibodies modified according to the present invention areadminister to the patient in one or more doses of substantially lessthan 375 mg/m² of the antibody, including but not limited to, where thedose is in the range from about 20 mg/m² to about 250 mg/m², or fromabout 50 mg/m² to about 200 mg/m². In another embodiment, the modifiedantibodies are used in a therapeutically effective amount from about 375mg/m² to about 1000 mg/m².

According to the invention one or more initial dose(s) of the antibodyfollowed by one or more subsequent dose(s) are administered, wherein themg/m² dose of the antibody in the subsequent dose(s) exceeds the mg/m²dose of the antibody in the initial dose(s). For example, the initialdose may be in the range from about 20 mg/m² to about 250 mg/m² and thesubsequent dose may be in the range from about 250 mg/m² to about 1000mg/m².

As noted herein, however, these suggested amounts of modified antibodyare subject to a great deal of therapeutic discretion. The key factor inselecting an appropriate dose and scheduling is the result obtained, asindicated herein. For example, relatively higher doses may be neededinitially for the treatment of ongoing and acute diseases. To obtain themost efficacious results, depending on the disease or disorder, theantagonist is administered as close to the first sign, diagnosis,appearance, or occurrence of the disease or disorder as possible orduring remissions of the disease or disorder.

The modified antibody of the present invention is administered by anysuitable means, including parenteral, subcutaneous, intraperitoneal,intrapulinonary, and intranasal, and, if desired for localimmunosuppressive treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, theantagonist may suitably be administered by pulse infusion, e.g., withdeclining doses of the antagonist. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.

According to the invention other compounds, such as cytotoxic agents,chemotherapeutic agents, immunosuppressive agents and/or cytokines areadministered with the antagonists herein. The combined administrationincludes coadministration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities.

It would be clear that the dose of the composition of the inventionrequired to achieve cures may be further reduced with scheduleoptimization.

In accordance with the practice of the invention, the pharmaceuticalcarrier may be a lipid carrier. The lipid carrier may be a phospholipid.Further, the lipid carrier may be a fatty acid.

Also, the lipid carrier may be a detergent. As used herein, a detergentis any substance that alters the surface tension of a liquid, generallylowering it.

In one example of the invention, the detergent may be a nonionicdetergent. Examples of nonionic detergents include, but are not limitedto, polysorbate 80 (also known as Tween 80 or polyoxyethylenesorbitanmonooleate), Brij, and Triton (for example Triton WR-1339 and TritonA-20).

Alternatively, the detergent may be an ionic detergent.

Additionally, in accordance with the invention, the lipid carrier may bea liposome. As used in this application, a “liposome” is any membranebound vesicle which contains any molecules of the invention orcombinations thereof.

EXEMPLARY EMBODIMENTS

Antibodies according to the invention comprise a variant heavy chainregion comprising at least one amino acid substitution relative to theparent non-substituted heavy chain region, leading to strongly reducedor abolished ADCC and CDC function, residual ADCP function, reduced orabolished binding to Fc receptors, reduced or abolished binding to C1qand reduced or abolished toxicities. Accordingly, provided are exemplaryembodiments as follows.

1. An antibody comprising a variant heavy chain region comprising atleast one amino acid substitution relative to the parent non-substitutedheavy chain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residue Asn297 and/orPro329, and wherein said substitution is Asn297Asp and/or Pro329Gly.2. The antibody according to embodiment 1, wherein induction of effectorfunction is reduced compared to effector function induced by an antibodycomprising the parent non-substituted heavy chain region.3. The antibody according to any one of claim 1 or 2, wherein theantibody is an IgG1 antibody.4. The antibody according to any one of embodiments 1 to 3, wherein theparent non-substituted antibody is an anti-CD20 antibody.5. The antibody according to any one of embodiments 1 to 4, wherein theparent non-substituted antibody is a type I anti-CD20 antibody.6. The antibody according to any one of embodiments 1 to 4, wherein theparent non-substituted antibody is a type II anti-CD20 antibody.7. The antibody according to any one of embodiments 1 to 4 or 6, whereinsaid parent non-substituted antibody is obinutuzumab.8. The antibody according to any one of embodiments 1 to 5, wherein saidparent non-substituted antibody is rituximab.9. The antibody according to any one of embodiments 1 to 8, whereinFcγRIII binding by the antibody comprising the variant heavy chainregion is abolished compared to binding to FcγRIII by the parentnon-substituted antibody comprising asparagine at position 297 orproline at position 329.10. The antibody according to any one of embodiments 1 to 9, whereinADCC function induced by the antibody comprising the variant heavy chainregion is abolished or strongly reduced compared to ADCC functioninduced by the parent non-substituted antibody comprising asparagine atposition 297 or proline at position 329.11. The antibody according to any one of embodiments 1 to 10, whereinFcγRI binding by the antibody comprising the variant heavy chain regionis reduced compared to binding to FcγRI by the parent non-substitutedantibody comprising asparagine at position 297 or proline at position329.12. The antibody according to any one of embodiments 1 to 11, whereinADCP function induced by the antibody comprising the variant heavy chainregion is reduced compared to ADCP function induced by the parentnon-substituted antibody comprising asparagine at position 297 orproline at position 329, wherein the antibody comprising the variantheavy chain retains residual ADCP function.13. The antibody according to any one of embodiments 1 to 12, whereinthe variant heavy chain region comprises a further amino acidsubstitution relative to the parent non-substituted heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residue Pro151, and wherein said furthersubstitution is at said amino acid residue Pro151, wherein direct celldeath induced by the antibody comprising the variant heavy chain regionis altered compared to direct cell death induced by the antibodycomprising proline at position 151.14. The antibody according to embodiment 13, wherein direct cell deathinduced by the antibody comprising the variant heavy chain region isincreased compared to direct cell death induced by the antibodycomprising proline at position 151.15. The antibody according to embodiment 13, wherein direct cell deathinduced by the antibody comprising the variant heavy chain region isdecreased compared to direct cell death induced by the antibodycomprising proline at position 151.16. The antibody according to embodiment 13, wherein Pro151 issubstituted with an amino acid selected from the group consisting ofalanine and phenylalanine.17. The antibody according to any one of embodiments 13 or 14, whereinPro151 is substituted with phenylalanine.18. The antibody according to any one of embodiments 13 or 15, whereinPro151 is substituted with alanine.19. The antibody according to any one of embodiments 1 to 18, whereinthe variant heavy chain region comprises a further amino acidsubstitution relative to the parent non-substituted heavy chain region,wherein the heavy chain region of the parent non-substituted antibodycomprises the amino acid residue Val11, and wherein said furthersubstitution is at said amino acid residue Val11, wherein direct celldeath induced by the antibody comprising the variant heavy chain regionis altered compared to direct cell death induced by the antibodycomprising valine at position 11.20. The antibody according to embodiment 19, wherein direct cell deathinduced by the antibody comprising the variant heavy chain region isincreased compared to direct cell death induced by the antibodycomprising valine at position 11.21. The antibody according to embodiment 19, wherein direct cell deathinduced by the antibody comprising the variant heavy chain region isdecreased compared to direct cell death induced by the antibodycomprising valine at position 11.22. The antibody according to embodiment 19, wherein Val11 issubstituted with an amino acid selected from the group consisting ofalanine, glycine, phenylalanine, threonine and tryptophan.23. The antibody according to any one of embodiments 19 or 20, whereinVal11 is substituted with an amino acid selected from the groupconsisting of phenylalanine, threonine and tryptophan.24. The antibody according to any one of embodiments 19 or 21, whereinVal11 is substituted with an amino acid selected from the groupconsisting of alanine and glycine.25. The antibody according to any one of embodiments 1 to 24, whereinthe antibody specifically binds to CD20.26. The antibody according to any one of embodiments 1 to 25, whereinthe antibody binds to CD20 with a dissociation constant (Kd) on cells of10 nM or less as determined by scatchard analysis.27. A polynucleotide encoding a variant heavy chain region of anantibody of any one of embodiments 1 to 26.28. A polynucleotide encoding a light chain region of an antibody of anyone of embodiments 1 to 26.29. A vector comprising at least one of the polynucleotides according tothe embodiments 27 and 28.30. The vector of embodiment 29 which is polycistronic.31. A host cell comprising one of the vectors according to embodiments29 and 30 or at least one of the polynucleotides according to theembodiments 27 and 28.32. A method for the production of an antibody according to any one ofembodiments 1 to 26 comprising (i) culturing the host cell of embodiment30 under conditions permitting the expression of said at least onepolynucleotide; and (ii) recovering said antibody from the culturemedium.33. A pharmaceutical composition comprising an antibody according to anyone of embodiments 1 to 26 and a pharmaceutically acceptable carrier.34. An antibody according to any one of embodiments 1 to 26 for use as amedicament.35. An antibody according to any one of embodiments 1 to 26 for use intreating a disease selected from the group consisting of proliferativedisorder and autoimmune disease.36. An antibody for use according to embodiment 35, characterized inthat said proliferative disorder is a CD20 expressing cancer.37. An antibody for use according to embodiment 36, characterized inthat said cancer is selected from the group consisting of lymphoma andlymphocytic leukemia.38. An antibody for use according to embodiment 35, characterized inthat said autoimmune disease is selected from the group consisting ofrheumatoid arthritis, lupus, multiple sclerosis, Sjögren's syndrome andtransplant rejection.39. A method for treating a disease selected from the group consistingof proliferative disorder and autoimmune disease comprisingadministering to an individual an effective amount of the antibodyaccording to any one of embodiments 1 to 26.40. The method according to embodiment 39, characterized in that saidproliferative disorder is a CD20 expressing cancer.41. The method according to embodiment 40, characterized in that saidcancer is selected from the group consisting of lymphoma and lymphocyticleukemia.42. The method according to embodiment 39, characterized in that saidautoimmune disease is selected from the group consisting of rheumatoidarthritis, lupus, multiple sclerosis, Sjögren's syndrome and transplantrejection.43. Use of the antibody according to any one of embodiments 1 to 26 forthe manufacture of a medicament.44. The use of embodiment 43, wherein the medicament is for treatment ofa disease selected from the group consisting of proliferative disorderand autoimmune disease.45. The use of embodiment 44, characterized in that said proliferativedisorder is a CD20 expressing cancer.46. The use of embodiment 45, characterized in that said cancer isselected from the group consisting of lymphoma and lymphocytic leukemia.47. The use of embodiment 44, characterized in that said autoimmunedisease is selected from the group consisting of rheumatoid arthritis,lupus, multiple sclerosis, Sjorgen's syndrome and transplant rejection.

The examples below explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those disclosed herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1

Antibodies

For the experiments described below antibodies against CD20(obinutuzumab (GA101), recommended INN, WHO Drug Information, Vol. 26,No. 4, 2012, p. 453 and rituximab, U.S. Pat. No. 7,381,560 andEP2000149B1) were used. All variants disclosed herein, e.g.,obinutuzumab P329G L234A L235A, obinutuzumab N297D, rituximab P329GL234A L235A and rituximab N297D were generated using PCR basedmutagenesis. IgG molecules were expressed in the HEK-EBNA or HEK293system, and purified using protein A and size exclusion chromatography.

Example 2 Cell Death Induction of Tumor Targets by Obinutuzumab andRituximab Fc Variants

The induction of cell death by obinutuzumab and rituximab variants wastested using CD20-expressing mantle cell lymphoma (Z-138). Briefly,cells were harvested, counted, checked for viability and re-suspended at0.526×10⁶ cells/ml in RPMI1640+10% FCS+1% Glutamax. 190 μl of cellsuspension (containing 0.1×10⁶ cells) were incubated in round-bottom96-well plate for 20 hours to 24 hours at 37° C. and 5% CO₂ in the cellincubator with different concentrations of the obinutuzumab or rituximabFc variants (16 ng/ml-10 μg/ml). The final volume was 200 μl per well.Afterwards, the cells were washed once with Annexin V Binding Buffer (10mM HEPES/NAOH pH7.4, 140 mM NaCl, 2.5 mM CaCl₂)) before incubation for30 min at 4° C. in the dark with 100 μl/well Annexin V FLUOS (Roche#11828681001, pre-diluted in Annexin V Binding Buffer 1:75). The cellswere washed by addition of 80 μl/well Annexin V Binding Buffer andimmediately analyzed by FACS using a FACS CantoII (Software FACS Diva)after addition of pre-diluted PI solution (Sigma Aldrich #P4864,1:4000).

FIG. 1 shows the induction of Phosphatidylserine surface expression onZ-138 as measured by Annexin V binding as well as PI staining in thepresence of different obinutuzumab or rituximab Fc variants. Allobinutuzumab variants induced significant cell death of Z-138 during 21hours incubation whereas all rituximab variants hardly induced celldeath under the chosen conditions. The induction of cell death wasindependent of the Fc part of the antibody. Only at high antibodyconcentrations obinutuzumab P329G L234A L235A and obinutuzumab N297Dseemed slightly inferior to obinutuzumab WT and GE.

Example 3 ADCC Induction by Obinutuzumab and Rituximab Fc Variants

The lysis of CD20-expressing tumor cells and subsequent NK cellactivation mediated by obinutuzumab and rituximab Fc variants wasassessed on Z-138 cells (mantle cell lymphoma) and SU-DHL-4 (B-NH).Human PBMCs were used as effectors and tumor cell lysis was detectedafter 4 hours of incubation with the different anti-CD20 antibodies.Briefly, target cells were harvested, washed, and plated at density of30 000 cells/well using round-bottom 96-well plates. Peripheral bloodmononuclear cells (PBMCs) were prepared by Histopaque densitycentrifugation of fresh blood obtained from healthy human donors. Freshblood was diluted with sterile PBS and layered over Histopaque gradient(Sigma, #10771). After centrifugation (450×g, 30 min, room temperature,no brake), the plasma above the PBMC-containing interphase was discardedand PBMCs transferred in a new falcon tube subsequently filled with 50ml of PBS. The mixture was centrifuged (350×g, 10 min, roomtemperature), the supernatant discarded and the PBMC pellet washed twicewith sterile PBS (centrifugation steps 300×g, 10 min). The resultingPBMC population was counted automatically (ViCell) and re-suspended inAIM V at 12.5 or 15×10⁶ cells/ml. For the ADCC, the antibodies wereadded at the indicated concentrations (range of 0.01 ng/ml-1000 ng/ml intriplicates). Furthermore, anti-CD107a (PE anti-human CD107a, Biolegend#328608) was added directly into the assay. PBMCs were added to targetcells at final E:T ratio of 21:1 or 25:1. Tumor cell lysis was assessedafter 4 hours of incubation at 37° C. and 5% CO₂ by quantification ofLDH released into cell supernatants by apoptotic/necrotic cells (LDHdetection kit, Roche Applied Science, #11 644 793 001). Maximal lysis ofthe target cells (=100%) was achieved by incubation of target cells with1% Triton X-100. Minimal lysis (=0%) refers to target cells co-incubatedwith effector cells without antibodies. For the assessment of NK cellactivation occurring upon ADCC, cells were centrifuged at 400×g for 4min and washed twice with FACS Buffer (PBS containing 2% FCS+5 mMEDTA+0.25% sodium acide). Surface staining for CD3 (PECy7 anti-humanCD3, Biolegend #300420) and CD56 (APC anti-human CD56, Biolegend#318310) was performed according to the suppliers' indications. Cellswere washed twice with 150 FACS Buffer and fixed using 150 FACS LysingSolution (BD #349202). Samples were analyzed using a BD FACS Cantoll.

FIGS. 2A and 2B show that obinutuzumab and rituximab GE antibodiesinduced a strong and target-specific killing of CD20+ target cells.Obinutuzumab GE was superior to rituximab GE and both antibodies weresuperior to the WT Fc variants of obinutuzumab and rituximab. Also as WTFc variants, obinutuzumab was superior to rituximab. Neither N297D norP329G L234A L235A Fc variants of obinutuzumab or rituximab induceddetectable tumor cell lysis using Z-138 or SU-DHL-4 as targets. Analysisof NK cell degranulation by flow cytometry confirmed the data andactivity of the tested Fc variants (FIGS. 2C and 2D). Obinutuzumab andrituximab GE antibodies induced strong degranulation as measured byCD107a expression on NK cells. Obinutuzumab GE was superior to rituximabGE and both antibodies were superior to the WT Fc variants ofobinutuzumab and rituximab. Also as WT Fc variants, obinutuzumab wassuperior to rituximab. Neither N297D nor P329G L234A L235A Fc variantsof obinutuzumab or rituximab induced detectable CD107a expression.

Example 4 CDC Induction by Obinutuzumab and Rituximab Fc Variants

The induction of CDC-dependent lysis of CD20-expressing tumor cellsmediated by obinutuzumab and rituximab Fc variants was assessed on Z-138cells and SU-DHL-4. Rabbit complement (Low-Tox Rabbit Complement,Cedarlane Laboratories Limited # CL3051) were used as complement andtumor cell lysis was detected after 2 hours or 24 hours of incubationwith the different anti-CD20 antibodies. Briefly, target cells wereharvested, washed, and plated at density of 50 000 cells/well usinground-bottom 96-well plates. For the CDC, the antibodies were added atthe indicated concentrations (0.01 μg/ml-100 μg/ml in triplicates) in 50μl/well and incubated with the cells for 10 min at RT. Meanwhile, thecomplement was dissolved in 1 ml Cedarlane Cytotoxicity Medium(Cedarlane Laboratories Limited # CL95100) and 2 ml AIM V per vial. 50μl/well prepared complement was added to the cells. Tumor cell lysis wasassessed after 2 hours of incubation at 37° C. and 5% CO₂ byquantification of LDH released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). After addition of 35 μl/well AlamarBlue (Biosource #DAL1100) the cells were incubated for further 22 hours at 37° C. and 5%CO₂. Tumor cell viability was measured using a Wallac Victor3 1420Multilabel Counter (excitation 584 nm, emission 612 nM). Maximal lysisof the target cells (=100%) was achieved by incubation of target cellswith 1% Triton X-100. Minimal lysis (=0%) refers to target cellsco-incubated with CDC without antibodies.

FIGS. 3A-3D show that rituximab GE and WT antibodies induced a strongand target-specific killing of CD20+ target cells after 2 hours and 22hours. Obinutuzumab GE and WT Fc variants also induced CDC but weresignificantly inferior to rituximab GE and WT. Neither N297D nor P329GLALA Fc variants of obinutuzumab or rituximab induced detectable tumorcell lysis using Z-138 or SU-DHL-4 as targets.

Example 5 B Cell Depletion in Human Whole Blood Mediated by Obinutuzumabor Rituximab Fc Variants

Normal B cell depletion mediated by obinutuzumab or rituximab Fcvariants was also assessed using fresh heparinized human blood fromhealthy volunteers. Briefly, fresh blood was collected inheparin-containing syringes. Blood aliquots (190 μL/well) were placed in96-deep well plates, supplemented with obinutuzumab or rituximab IgGvariants dilutions (10 μL/well ranging from 0.1 ng/ml up to 1000 ng/ml)and incubated for 20 hours to 24 hours up at 37° C. and 5% CO₂ in ahumidified cell incubator. After incubation, blood was mixed bypipetting up and down before 35 μL/well blood aliquots were transferredin 96-round-bottom plates and incubated with fluorescent anti-CD45 (APCanti-human CD45, BD #555485), anti-CD19 (PE Anti-human CD19, Biolegend#302208) and anti-CD3 (PE/Cy7Anti-human CD3, Biolegend #300420) in total55 μL volume for flow cytometry. After 15 min incubation at roomtemperature (in the dark) 200 μL/well of FACS lysis solution (BD#349202) was added to deplete erythrocytes and to fix cells prior toflow cytometry using a BD FACSCantoII.

FIG. 4A (Donor 1) and FIG. 4B (Donor 2) show that obinutuzumab andrituximab GE antibodies induced a strong killing of CD20+/CD19+ B cellsin healthy human whole blood. Obinutuzumab GE was superior to rituximabGE and both antibodies were superior to the WT Fc variants ofobinutuzumab and rituximab. Also as WT Fc variants, obinutuzumab wassuperior to rituximab. Obinutuzumab N297D and P329G L234A L235A Fcvariants also induced some B cell depletion which is due to theFc-independent direct cell death effect mediated by obinutuzumab. Incontrast to that, rituximab N297D and P329G L234A L235A Fc variants didnot induce detectable B cell depletion. As type I CD20 binder and asshown in example 2, rituximab induced only minor direct cell deathinduction compared to obinutuzumab. Table 2 shows the corresponding EC50values calculated with GraphPad Prism.

TABLE 2 EC50 (ng/ml) of B cell depletion EC50 EC50 (ng/ml)_donor 1(ng/ml)_donor 2 obinutuzumab GE 0.4 2.8 obinutuzumab WT 6.4 34.8obinutuzumab P329G 91.0 120.5 L234A L235A obinutuzumab N297D 126.8 166.2rituximab GE 5.4 23.9 rituximab VVT 14.1 40.8 rituximab P329G n.d. n.d.L234A L235A rituximab N297D n.d. n.d.

Example 6 ADCP Induction by Obinutuzumab and Rituximab Fc Variants

The antibody-dependent phagocytosis (ADCP) of CD20-expressing tumorcells by monocyte-derived macrophages (M1 or M2c) mediated byobinutuzumab and rituximab Fc variants was assessed on Z-138 cells(mantle cell lymphoma) and SU-DHL-4 (B-NH). Pan monocytes were isolatedfrom human PBMCs (PBMC isolation see example 3) and differentiated intomacrophages using 30 ng/ml M-CSF for 7 days followed by 2 day incubationwith 10 ng/ml IL-10 to generate M2c macrophages or 1 day incubation with100 ng/ml IFNg+100 ng/ml LPS to generate M1 macrophages. DifferentiatedM1 and M2c macrophages were labeled with PKH26 (Sigma Aldrich) accordingto the manufacturer's instructions and used as effectors andphagocytosis was detected after 4 h of incubation with differentanti-CD20 antibodies. Briefly, target cells were harvested, washed, andlabeled with 2 μM CFSE (Sigma Aldrich) prior to plating at a density of30 000 cells/well using 96-well UpCell plates. For ADCP, the antibodieswere added at 50 ng/ml or 1000 ng/ml in triplicates in the presence orabsence of 10 mg/ml Redimune (Behring). PKH26-labeled M1 or M2cmacrophages were added to target cells at final E:T ratio of 3:1. ADCPwas assessed after 4 hours of incubation at 37° C. and 5% CO₂ byanalyzing the CFSE-PKH26-double positive cells in flow cytometry using aBD FACS Cantoll.

FIGS. 5A-5D show the ADCP after 4 hours incubation of SU-DHL-4 or Z-138with M1 or M2c macrophages in the presence of 10 mg/ml unspecific humanIgGs (Redimune). M1 and M2c macrophages performed comparablephagocytosis under the chosen conditions. After 4 hours, phagocytosis ofSUDHL4 was significantly higher in the presence of 1000 ng/ml anti-CD20abs compared to Z-138. Obinutuzumab WT and N297D induced significant andcomparable phagocytosis using SU-DHL-4 and Z-138. Obinutuzumab GEinduced slightly stronger phagocytosis mainly of CD20+SU-DHL-4 targetcells using M2c as effectors compared to obinutuzumab WT and N297D inthe presence of 10 mg/ml Redimune.

Example 7 Antitumor Activity of a Type II Anti-CD20 AntibodyObinutuzumab (GA101), a Type I Anti-CD20 Antibody Rituximab and theObinutuzumab Variants GA101 P329G L234A L235A and GA101 Aglyco (N297D)Test Agents

The antibodies were provided as stock solution from Roche Glycart AG,Schlieren, Switzerland, in histidine buffer. The antibody was dilutedwith 0.9% NaCl solution prior to in-vivo application.

Cell Lines and Culture Conditions

The human SU-DHL-4 lymphoma cell line was cultured in RPMI 1640supplemented with 10% fetal bovine serum (PAA Laboratories, Austria) and2 mM L-glutamine at 37° C. in a water-saturated atmosphere at 5% CO₂.For in-vivo xenograft experiments the cells were co-injected withMatrigel.

Animals

Female SCID beige mice, age 5 to 6 weeks at arrival (purchased fromCharles River, Sulzfeld, Germany), were maintained in the quarantinepart of the animal facility for one week and afterwards underspecific-pathogen-free condition with daily cycles of 12 hours light/12hours darkness according to guidelines (GV-Solas; Felasa; TierschG). Theexperimental study protocol was reviewed and approved by Roche and thelocal government (Regierung von Oberbayern; registration no.55.2-1-54-2531.2-26-09). Diet food (KLIBA NAFAG 3807) and water(filtered) were provided ad libitum.

Induction of SC SU-DHL-4 Tumors in SCID Beige Mice

Five millions (5×10⁶) SU-DHL-4 tumor cells in 100 μl of PBS withmatrigel (50:50, BD Biosciences, France) were subcutaneously (SC)injected into the right flank of female SCID beige mice.

Monitoring

Animals were monitored daily for clinical symptoms and detection ofadverse effects. During the experiment the body weight of animals waschecked two times a week and tumor volume was measured by caliper.

Treatment of Animals

Animal treatment started at the day of randomization 21 days after tumorcell inoculation. Humanized type II anti-CD20 antibody obinutuzumab(GA101), rituximab, GA101 P329G L234A L235A and obinutuzumab N297D wasadministered as single agent i.p. q7d once weekly (day 21, 28, 35 and42) for 4 weeks at a dosage of 30 mg/kg. The corresponding vehicle wasadministered on the same days.

Tumor Growth Inhibition (TGI) on Day 49

Monotherapy treatment using GA101 P329G L234A L235A, rituximab orobinutuzumab N297D resulted in tumor growth inhibition of 62%, 71% or93%, respectively (based on medians). Obinutuzumab treatment showedtumor regression (TGI>100%) on day 49 after tumor cell inoculation.

Nonparametric Treatment-to-Control-Ratios (TCRnpar) on Day 49

Nonparametric Treatment-to-Control-Ratios (TCRnpar) and the two-sidednonparametric confidence intervals (CI) was calculated based on baselinecorrected data by ratio to assess statistical significance on day 49after tumor cell inoculation. Each treatment was statisticallysignificant compared to the control group.

TABLE 3 Summary of results according to FIG. 6 Tumor free TGI np TCR[95% CI] Animals Treatment schedule (%) compared to vehicle on Day 49Vehicle — — [—] 0 obinutuzumab (GA101) >100 0 [0-0] 9 30 mg/kg; q7dx4;IP GA101 P329G LALA 62 0.43 [0.31-0.63] 0 30 mg/kg; q7dx4; IP GA101N297D 30 mg/kg; q7dx4; IP 93 0.14 [0-0.35] 2 rituximab 71 0.37[0.29-0.54] 0 30 mg/kg; q7dx4; IP

Example 8 FcγRI SPR Assay

The SPR interaction analysis of captured FcγRI and IgG1 Fc variants wasperformed on a

Biacore T100 system (GE Healthcare) with high immobilized anti-Hiscapturing antibody (GE Healthcare). Immobilization of the anti-Hiscapturing antibody was performed on a CMS chip using the standard aminecoupling kit (GE Healthcare) at pH 4.5 10 mM sodium acetate and 10 μg/mlanti-His solution. The immobilization level of anti-His reached morethan 10.000 RU. FcγRI was applied in a 100 nM solution, in the runningbuffer HBS-P+ and captured with a pulse of 60 sec at a flow rate of 30μl per min. Subsequently GA101 N297D was applied in a serial dilutionfrom 1000-62.5 nM in HBS-P+ and a flow rate of 30 μl per min for 60 sec.The dissociation phase was monitored for 180 sec. The surface wasregenerated by a 60 sec washing step with a 10 mM Glycine pH 1.5 at aflow rate of 30 μl per min. The stabilization period took 60 sec. TheBiacore T100 evaluation software was used for data analysis.

FcγRI SPR Assay Steady State Evaluation

For the steady state affinity concentration determination the responsevalues from the end of association were used to plot the responseagainst the concentration. The Biacore T100 evaluation software was usedto calculate the affinity of samples for immobilized FcγRI receptor fromthe plot.

The interaction measurement of GA101 N297D to FcγRI in comparison to awild type IgG1 (Herceptin WT) molecule and a IgG4SPLE molecule(P-Selectin), demonstrated a clear binding order (FIGS. 7A and 7B). TheIgG1 WT displayed the highest interaction signal of up to 250 ResponseUnits (RU), the GA101 N297D displayed a RU signal of 150, whereas theP-Selectin displayed only a very low binding signal of 25 ResponseUnits, measured at 200 nM antibody concentration (FIG. 7C). It is knownfor IgG1 Wt antibodies to bind FcγRI with an affinity in the nanomolarrange. To determine the GA101 N297D affinity a steady state affinitydetermination was performed. The affinity was measured as 234 nM (FIGS.7D and 7E), which is roughly 50 fold below the IgG1 Wt affinity, butsignificant stronger compared with the IgG4SPLE FcγRI binding reduction,where it is not possible to determine a certain affinity.

Example 9 FcγRIII SPR Assay

The SPR interaction analysis of captured FcγRIIIA and IgG1 Fc variantswas performed on a Biacore T100 system (GE Healthcare) with highimmobilized anti-His capturing antibody (GE Healthcare). Immobilizationof the anti-His capturing antibody was performed on a CMS chip using thestandard amine coupling kit (GE Healthcare) at pH 4.5 10 mM sodiumacetate and 10 μg/ml anti-His solution. The immobilization level ofanti-His reached more than 10.000 RU. FcγRIIIA was applied in a 200 nMsolution, in the running buffer HBS-P+ and captured with a pulse of 60sec at a flow rate of 30 μl per min. Subsequently GA101 N297D wasapplied in a serial dilution from 1000 to 62.5 nM in HBS-P+ and a flowrate of 30 μl per min for 60 sec. The dissociation phase was monitoredfor 180 sec. The surface was regenerated by a 60 sec washing step with a10 mM Glycine pH 1.5 at a flow rate of 30 μl per min. The stabilizationperiod took 60 sec. The Biacore T100 evaluation software was used fordata analysis.

The time resolved binding determination of GA101 N297D displayed nomeasurable interaction with FcγRIIIA, in contrast to the Herceptin WTIgG1 molecule (FIGS. 8A and 8B). Performing the analogous steady stateaffinity determination experiment no clear concentration dependentbinding signal could be measured. The highest concentration of 1 μMdisplayed only a RU value of 5 and no saturation of the interactioncould be observed (FIG. 8C). Consequently, the saturable interaction wasbelow the value that could be determined by SPR (high μM range)comparably to the affinity of the IgG4 SPLE control, which did also notdisplay a relevant interaction signal (FIG. 8D).

Example 10 SPR FcγR Capture Setup (FIGS. 7F, 8D, 9A, and 9B)

The SPR interaction analysis of captured FcγRs (FcγRIa, FcγRIIa (R131)and FcγRIIa (H131), FcγRIIIa (V158)) and IgG1 Fc variants was performedon a Biacore T200 system (GE Healthcare) with high immobilized anti-Hiscapturing antibody (GE Healthcare). Immobilization of the anti-Hiscapturing antibody was performed on a CMS chip using the standard aminecoupling kit (GE Healthcare) at pH 4.5 10 mM sodium acetate and 10 μg/mlanti-His solution. The immobilization level of anti-His reached >10 000RU. FcγRs were prepared as solution of 100 nM each, using running bufferHBS-P+ and were captured with a pulse of 60 s at a flow rate of 30μl/min. Subsequently, IgG1 Fc his-tagged variants were applied at aconcentration of 100 nM in HBS-P+ and a flow rate of 30 μl/min for 60 s.

The dissociation phase was monitored for 180 s. The surface wasregenerated by a 60 s washing step with a 10 mM Glycine pH 1.5 at a flowrate of 30 μl/min. The stabilization period was set to 60 s. The BiacoreT200 evaluation software was used for data analysis.

Example 11 Thermostability

The melting temperature, Tm, was assessed by recording the intrinsicTryptophan fluorescence with an Optim1000 instrument (Avacta AnalyticalInc.). Samples were prepared at approx. 1 mg/mL in 20 mM Histidinechloride, 140 mM NaCl, pH 6.0 and transferred to a 9 μL multi-cuvettearray. The multi-cuvette array was heated from 30° C. to 90° C. at aconstant rate of 0.1° C./minute. The instrument continuously recordsfluorescence emission spectra after excitation with a 266 nm laser,providing a data point approximately every 0.6° C. The meltingtemperature, Tm, is determined by plotting the fluorescence intensityagainst the temperature and Tm is defined as the inflection point inthese curves.

Both N297D variants display an aggregation temperature 6° C. lowercompared to both respective P329G variants (FIGS. 13A and 13B).Additionally, the melting temperature of the deglycosylated N297Dvariants is below the more stable P329G variants. The P329G stays in anactive confirmation at higher temperatures and displays thereby a betterstructural integrity.

1. An antibody comprising a variant heavy chain region comprising atleast one amino acid substitution relative to the parent non-substitutedheavy chain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residue Asn297 and/orPro329, and wherein said substitution is Asn297Asp and/or Pro329Gly,wherein the residues are numbered according to the EU index as in Kabat,wherein ADCP function induced by the antibody comprising the variantheavy chain region is reduced compared to ADCP function induced by theparent non-substituted antibody, wherein the antibody comprising thevariant heavy chain retains residual ADCP function.
 2. The antibodyaccording to claim 1, wherein induction of effector function is reducedcompared to effector function induced by an antibody comprising theparent non-substituted heavy chain region.
 3. The antibody according toclaim 1, wherein the antibody is an IgG1 antibody.
 4. The antibodyaccording to claim 1, wherein the parent non-substituted antibody isobinutuzumab or rituximab.
 5. The antibody according to claim 1, whereinFcγRIII binding by the antibody comprising the variant heavy chainregion is abolished compared to binding to FcγRIII by the parentnon-substituted antibody.
 6. The antibody according to claim 1, whereinADCC function induced by the antibody comprising the variant heavy chainregion is abolished or strongly reduced compared to ADCC functioninduced by the parent non-substituted antibody.
 7. The antibodyaccording to claim 1, wherein FcγRI binding by the antibody comprisingthe variant heavy chain region is reduced compared to binding to FcγRIby the parent non-substituted antibody.
 8. The antibody according toclaim 1, wherein the variant heavy chain region comprises a furtheramino acid substitution relative to the parent non-substituted heavychain region, wherein the heavy chain region of the parentnon-substituted antibody comprises the amino acid residue Pro151, andwherein said further substitution is at said amino acid residue Pro151,wherein direct cell death induced by the antibody comprising the variantheavy chain region is altered compared to direct cell death induced bythe antibody comprising proline at position
 151. 9. The antibodyaccording to claim 8, wherein direct cell death induced by the antibodycomprising the variant heavy chain region is increased compared todirect cell death induced by the antibody comprising proline at position151.
 10. The antibody according to claim 8, wherein Pro151 issubstituted with phenylalanine.
 11. The antibody according to claim 1,wherein the antibody specifically binds to CD20.
 12. A pharmaceuticalcomposition comprising an antibody according to claim 1 and apharmaceutically acceptable carrier. 13-15. (canceled)
 16. A method fortreating a disease selected from the group consisting of proliferativedisorder and autoimmune disease comprising administering to anindividual an effective amount of the antibody according to claim
 1. 17.The method according to claim 16, characterized in that saidproliferative disorder is a CD20 expressing cancer.
 18. (canceled) 19.(canceled)